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Cited2 controls left-right patterning and heart development through a Nodal-Pitx2c pathway

Nature Genetics volume 36, pages 1189–1196 (2004)Cite this article

Abstract

Malformations of the septum, outflow tract and aortic arch are the most common congenital cardiovascular defects and occur in mice lacking Cited2, a transcriptional coactivator of TFAP2. Here we show that Cited2−/− mice also develop laterality defects, including right isomerism, abnormal cardiac looping and hyposplenia, which are suppressed on a mixed genetic background. Cited2−/− mice lack expression of the Nodal target genes Pitx2c, Nodal and Ebaf in the left lateral plate mesoderm, where they are required for establishing laterality and cardiovascular development. CITED2 and TFAP2 were detected at the Pitx2c promoter in embryonic hearts, and they activate Pitx2c transcription in transient transfection assays. We propose that an abnormal Nodal-Pitx2c pathway represents a unifying mechanism for the cardiovascular malformations observed in Cited2−/− mice, and that such malformations may be the sole manifestation of a laterality defect.

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Figure 1: Cardiovascular laterality defects in Cited2−/− embryos at 15.5 d.p.c.
Figure 2: Noncardiovascular laterality defects in Cited2−/− embryos.
Figure 3: Hand1 and Pitx2 expression in Cited2−/− embryos.
Figure 4: Cardiac and laterality defects in mice lacking Pitx2c.
Figure 5: Cited2 is required for Nodal-activated gene transcription.
Figure 6: Evolutionary conservation of the Pitx2c P1 promoter.
Figure 7: CITED2 and TFAP2 control Pitx2c expression.

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References

  1. Bruneau, B.G. Transcriptional regulation of vertebrate cardiac morphogenesis. Circ. Res. 90, 509–519 (2002).

    Article  Google Scholar 

  2. Goodman, R.H. & Smolik, S. CBP/p300 in cell growth, transformation, and development. Genes Dev. 14, 1553–1577 (2000).

    CAS  PubMed  Google Scholar 

  3. Petrij, F. et al. Rubinstein-Taybi syndrome caused by mutations in the transcriptional co-activator CBP. Nature 376, 348–351 (1995).

    Article  CAS  Google Scholar 

  4. Stevens, C.A. & Bhakta, M.G. Cardiac abnormalities in the Rubinstein-Taybi syndrome. Am. J. Med. Genet. 59, 346–348 (1995).

    Article  CAS  Google Scholar 

  5. Bhattacharya, S. et al. Functional role of p35srj, a novel p300/CBP binding protein, during transactivation by HIF-1. Genes Dev. 13, 64–75 (1999).

    Article  CAS  Google Scholar 

  6. Dunwoodie, S.L., Rodriguez, T.A. & Beddington, R.S.P. Msg1 and Mrg1, founding members of a gene family, show distinct patterns of gene, expression during mouse embryogenesis. Mech. Dev. 72, 27–40 (1998).

    Article  CAS  Google Scholar 

  7. Sun, H.B., Zhu, Y.X., Yin, T., Sledge, G. & Yang, Y.C. MRG1, the product of a melanocyte-specific gene related gene, is a cytokine-inducible transcription factor with transformation activity. Proc. Natl. Acad. Sci. USA 95, 13555–13560 (1998).

    Article  CAS  Google Scholar 

  8. Freedman, S.J. et al. Structural basis for negative regulation of hypoxia-inducible factor-1α by CITED2. Nat. Struct. Biol. 10, 504–512 (2003).

    Article  CAS  Google Scholar 

  9. Bamforth, S.D. et al. Cardiac malformations, adrenal agenesis, neural crest defects and exencephaly in mice lacking Cited2, a new Tfap2 co-activator. Nat. Genet. 29, 469–474 (2001).

    Article  CAS  Google Scholar 

  10. Barbera, J.P. et al. Folic acid prevents exencephaly in Cited2 deficient mice. Hum. Mol. Genet. 11, 283–293 (2002).

    Article  CAS  Google Scholar 

  11. Weninger, W.J. & Mohun, T. Phenotyping transgenic embryos: a rapid 3-D screening method based on episcopic fluorescence image capturing. Nat. Genet. 30, 59–65 (2002).

    Article  CAS  Google Scholar 

  12. Yin, Z. et al. The essential role of Cited2, a negative regulator for HIF-1α, in heart development and neurulation. Proc. Natl. Acad. Sci. USA 99, 10488–10493 (2002).

    Article  CAS  Google Scholar 

  13. Schneider, J.E. et al. Rapid identification and 3D reconstruction of complex cardiac malformations in transgenic mouse embryos using fast gradient echo sequence magnetic resonance imaging. J. Mol. Cell. Cardiol. 35, 217–222 (2003).

    Article  CAS  Google Scholar 

  14. Kranc, K.R. et al. Transcriptional Coactivator Cited2 Induces Bmi1 and Mel18 and Controls Fibroblast Proliferation via Ink4a/ARF. Mol. Cell. Biol. 23, 7658–7666 (2003).

    Article  CAS  Google Scholar 

  15. Braganca, J. et al. Physical and Functional Interactions among AP-2 Transcription Factors, p300/CREB-binding Protein, and CITED2. J. Biol. Chem. 278, 16021–16029 (2003).

    Article  CAS  Google Scholar 

  16. Glenn, D.J. & Maurer, R.A. MRG1 Binds to the LIM Domain of Lhx2 and may function as a coactivator to stimulate glycoprotein hormone alpha-subunit gene expression. J. Biol. Chem. 274, 36159–36167 (1999).

    Article  CAS  Google Scholar 

  17. Brewer, S., Jiang, X., Donaldson, S., Williams, T. & Sucov, H.M. Requirement for AP-2α in cardiac outflow tract morphogenesis. Mech. Dev. 110, 139–149 (2002).

    Article  CAS  Google Scholar 

  18. Satoda, M. et al. Mutations in TFAP2B cause Char syndrome, a familial form of patent ductus arteriosus. Nat. Genet. 25, 42–46 (2000).

    Article  CAS  Google Scholar 

  19. Thomas, T., Yamagishi, H., Overbeek, P.A., Olson, E.N. & Srivastava, D. The bHLH factors, dHAND and eHAND, specify pulmonary and systemic cardiac ventricles independent of left-right sidedness. Dev. Biol. 196, 228–236 (1998).

    Article  CAS  Google Scholar 

  20. Gage, P.J., Suh, H. & Camper, S.A. Dosage requirement of Pitx2 for development of multiple organs. Development 126, 4643–4651 (1999).

    CAS  PubMed  Google Scholar 

  21. Kitamura, K. et al. Mouse Pitx2 deficiency leads to anomalies of the ventral body wall, heart, extra- and periocular mesoderm and right pulmonary isomerism. Development 126, 5749–5758 (1999).

    CAS  PubMed  Google Scholar 

  22. Lin, C.R. et al. Pitx2 regulates lung asymmetry cardiac positioning and pituitary and tooth morphogenesis. Nature 401, 279–282 (1999).

    Article  CAS  Google Scholar 

  23. Lu, M.F., Pressman, C., Dyer, R., Johnson, R.L. & Martin, J.F. Function of Rieger syndrome gene in left-right asymmetry and craniofacial development. Nature 401, 276–278 (1999).

    Article  CAS  Google Scholar 

  24. Liu, C., Liu, W., Lu, M.F., Brown, N.A. & Martin, J.F. Regulation of left-right asymmetry by thresholds of Pitx2c activity. Development 128, 2039–2048 (2001).

    CAS  PubMed  Google Scholar 

  25. Liu, C. et al. Pitx2c patterns anterior myocardium and aortic arch vessels and is required for local cell movement into atrioventricular cushions. Development 129, 5081–5091 (2002).

    Article  CAS  Google Scholar 

  26. Hamada, H., Meno, C., Watanabe, D. & Saijoh, Y. Establishment of vertebrate left-right asymmetry. Nat. Rev. Genet. 3, 103–113 (2002).

    Article  CAS  Google Scholar 

  27. Saijoh, Y., Oki, S., Ohishi, S. & Hamada, H. Left-right patterning of the mouse lateral plate requires nodal produced in the node. Dev. Biol. 256, 160–172 (2003).

    Article  CAS  Google Scholar 

  28. Meno, C. et al. Diffusion of nodal signaling activity in the absence of the feedback inhibitor Lefty2. Dev. Cell 1, 127–138 (2001).

    Article  CAS  Google Scholar 

  29. Saijoh, Y. et al. Left-right asymmetric expression of lefty2 and nodal is induced by a signaling pathway that includes the transcription factor FAST2. Mol. Cell 5, 35–47 (2000).

    Article  CAS  Google Scholar 

  30. Shen, M.M., Wang, H. & Leder, P. A differential display strategy identifies Cryptic, a novel EGF-related gene expressed in the axial and lateral mesoderm during mouse gastrulation. Development 124, 429–442 (1997).

    CAS  PubMed  Google Scholar 

  31. Yamamoto, M. et al. Nodal signaling induces the midline barrier by activating Nodal expression in the lateral plate. Development 130, 1795–1804 (2003).

    Article  CAS  Google Scholar 

  32. Shiratori, H. et al. Two-step regulation of left-right asymmetric expression of Pitx2: initiation by nodal signaling and maintenance by Nkx2. Mol. Cell 7, 137–149 (2001).

    Article  CAS  Google Scholar 

  33. Prestridge, D.S. SIGNAL SCAN: a computer program that scans DNA sequences for eukaryotic transcriptional elements. Comput. Appl. Biosci. 7, 203–206 (1991).

    CAS  PubMed  Google Scholar 

  34. Brennan, J., Norris, D.P. & Robertson, E.J. Nodal activity in the node governs left-right asymmetry. Genes Dev. 16, 2339–2344 (2002).

    Article  CAS  Google Scholar 

  35. Norris, D.P., Brennan, J., Bikoff, E.K. & Robertson, E.J. The Foxh1-dependent autoregulatory enhancer controls the level of Nodal signals in the mouse embryo. Development 129, 3455–3468 (2002).

    CAS  PubMed  Google Scholar 

  36. Yan, Y.T. et al. Conserved requirement for EGF-CFC genes in vertebrate left-right axis formation. Genes Dev. 13, 2527–2537 (1999).

    Article  CAS  Google Scholar 

  37. Gaio, U. et al. A role of the cryptic gene in the correct establishment of the left-right axis. Curr. Biol. 9, 1339–1342 (1999).

    Article  CAS  Google Scholar 

  38. Pena, P. et al. Activator protein-2 mediates transcriptional activation of the CYP11A1 gene by interaction with Sp1 rather than binding to DNA. Mol. Endocrinol. 13, 1402–1416 (1999).

    Article  CAS  Google Scholar 

  39. Waterston, R.H. et al. Initial sequencing and comparative analysis of the mouse genome. Nature 420, 520–562 (2002).

    Article  CAS  Google Scholar 

  40. Kent, G.C. & Carr, R.K. Comparative Anatomy of the Vertebrates 314–350 (McGraw Hill, Boston, 2001).

    Google Scholar 

  41. Clark, E.B. Etiology of congenital cardiac malformations: epidemiology and genetics. in Moss and Adams' Heart Disease in Infants, Children, and Adolescents (eds. Allen, H.D., Gutgessell, H.P., Clark, E.B. & Driscoll, D.J.) 64–79 (Lipincott Williams & Wilkins, Philadelphia, 2001).

    Google Scholar 

  42. Wilkinson, D.G. Whole mount in situ hybridization of vertebrate embryos. in In situ Hybridization (ed. Wilkinson, D.G.) 75–83 (IRL, Oxford, 1992).

    Google Scholar 

  43. Ryan, A.K. et al. Pitx2 determines left-right asymmetry of internal organs in vertebrates. Nature 394, 545–551 (1998).

    Article  CAS  Google Scholar 

  44. Tatusova, T.A. & Madden, T.L. BLAST 2 Sequences, a new tool for comparing protein and nucleotide sequences. FEMS Microbiol. Lett. 174, 247–250 (1999).

    Article  CAS  Google Scholar 

  45. Ausubel, F. et al. Short Protocols in Molecular Biology (John Wiley & Sons, 1995).

    Google Scholar 

  46. Boyd, K.E., Wells, J., Gutman, J., Bartley, S.M. & Farnham, P.J. c-Myc target gene specificity is determined by a post-DNA binding mechanism. Proc. Natl. Acad. Sci. USA 95, 13887–13892 (1998).

    Article  CAS  Google Scholar 

  47. Brown, N.A. & Anderson, R.H. Symmetry and laterality in the human heart: developmental implications. in Heart Development (eds. Harvey, R.P. & Rosenthal, A.) 447–462 (Academic, San Diego, 1999).

    Chapter  Google Scholar 

  48. Kaufman, M. & Bard, J.B.L. The Anatomical Basis of Mouse Development 33–46 (Academic, San Diego, 1999).

    Google Scholar 

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Acknowledgements

We thank H. Hamada, H. Shiratori, H. Hurst and P. Riley for reagents; H. Koseki and Y. Fujimura for ChIP protocols; and R. Copley for discussions. C.R.F. is a Wellcome Trust Prize Student, and S.B. a Wellcome Trust Senior Research Fellow. These studies were funded by the Wellcome Trust.

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Author notes

  1. Simon D Bamforth, José Bragança and Cassandra R Farthing: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cardiovascular Medicine, University of Oxford, Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford, OX3 7BN, UK

    Simon D Bamforth, José Bragança, Cassandra R Farthing, Jürgen E Schneider, Carol Broadbent, Anna C Michell, Stefan Neubauer & Shoumo Bhattacharya

  2. Department of Physiology, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK

    Kieran Clarke

  3. MRC Mammalian Genetics Unit, Harwell, OX11 0RD, UK

    Dominic Norris

  4. St. George's Hospital Medical School, University of London, Cranmer Terrace, London, SW17 0RE, UK

    Nigel A Brown

  5. Cardiac Unit, Institute of Child Health, University College London, 30 Guilford Street, London, WC1N 1EH, UK

    Robert H Anderson

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Correspondence to Shoumo Bhattacharya.

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Supplementary information

Supplementary Fig. 1

Pitx2c, Tcfap2, Cited2 and Actb (β-actin, control) expression was assessed using RT-PCR. (PDF 294 kb)

Supplementary Methods (PDF 6 kb)

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Bamforth, S., Bragança, J., Farthing, C. et al. Cited2 controls left-right patterning and heart development through a Nodal-Pitx2c pathway. Nat Genet 36, 1189–1196 (2004). https://doi.org/10.1038/ng1446

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