The Tgf- β3 null mutant mouse palate presents several cellular anomalies that lead to the appearance of cleft palate. One of them concerns the cell proliferation of both the palatal medial edge epithelium and mesenchyme. In this work, our aim was to determine whether there was any variation in the presence/distribution of several cell proliferation-related molecules that could be responsible for the cell proliferation defects observed in these palates. Our results showed no difference in the presence of EGF-R, PDGF-A, TGF-β2, Bmp-2, and Bmp-4, and differences were minimal for FGF-10 and Shh. However, the expression of EGF and Msx-1 changed substantially. The shift of the EGF protein expression was the one that most correlated with that of cell proliferation. This molecule is regulated by TGF-β3, and experiments blocking its activity in culture suggest that EGF misexpression in the Tgf- β3 null mutant mouse palate plays a role in the cell proliferation defect observed.

Keywords:
Tgf- β3, Knockout, Palate, Epidermal growth factor, Msx-1
1.
Abbott, B.D., D.S. Best, M.G. Narotsky (2005) Teratogenic effects of retinoic acid are modulated in mice lacking expression of epidermal growth factor and transforming growth factor-alpha. Birth Defects Res A Clin Mol Teratol 73: 204–217.
2.
Abbott, B.D., R.M. Pratt (1987) Retinoids and epidermal growth factor alter embryonic mouse palatal epithelial and mesenchymal cell differentiation in organ culture. J Craniofac Genet Dev Biol 7: 219–240.
3.
Abbott, B.D., R.M. Pratt (1988) EGF receptor expression in the developing tooth is altered by exogenous retinoic acid and EGF. Dev Biol 128: 300–304.
4.
Abud, H.E., N. Watson, J.K. Heath (2005) Growth of intestinal epithelium in organ culture is dependent on EGF signalling. Exp Cell Res 303: 252–262.
5.
Alappat, S.R., Z. Zhang, K. Suzuki, X. Zhang, H. Liu, R. Jiang, G. Yamada, Y. Chen (2005) The cellular and molecular etiology of the cleft secondary palate in Fgf10 mutant mice. Dev Biol 277: 102–113.
6.
Barlow, A.J., P.H. Francis-West (1997) Ectopic application of recombinant BMP-2 and BMP-4 can change patterning of developing chick facial primordia. Development 124: 391–398.
7.
Betsholtz, C., L. Karlsson, P. Lindahl (2001) Developmental roles of platelet-derived growth factors. Bioessays 23: 494–507.
8.
Bitgood, M.J., A.P. McMahon (1995) Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo. Dev Biol 172: 126–138.
9.
Blavier, L., A. Lazaryev, J. Groffen, N. Heisterkamp, Y.A. DeClerk, V. Kaartinen (2001) TGF-beta3-induced palatogenesis requires matrix metalloproteinases. Mol Biol Cell 12: 1457–1466.
10.
Brunet, C.L., P.M. Sharpe, M.W. Ferguson (1993) The distribution of epidermal growth factor binding sites in the developing mouse palate. Int J Dev Biol 37: 451–458.
11.
Bryant, P.L., J.E. Schmid, S.E. Fenton, A.R. Buckalew, B.D. Abbott (2001) Teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice lacking the expression of EGF and/or TGF-alpha. Toxicol Sci 62: 103–114.
12.
Burdett, D.N., J.D. Waterfield, R.M. Shah (1988) Vertical development of the secondary palate in hamster embryos following exposure to 6-mercaptopurine. Teratology 37: 591–597.
13.
Carette, M.J., M.W. Ferguson (1992) The fate of medial edge epithelial cells during palatal fusion in vitro: an analysis by DiI labelling and confocal microscopy. Development 114: 379–388.
14.
Citterio, H.L., D.A. Gaillard (1994) Expression of transforming growth factor alpha (TGF alpha), epidermal growth factor receptor (EGF-R) and cell proliferation during human palatogenesis: an immunohistochemical study. Int J Dev Biol 38: 499–505.
15.
Cobourne, M.T., G.M. Xavier, M. Depew, L. Hagan, J. Sealby, Z. Webster, P.T. Sharpe (2009) Sonic hedgehog signalling inhibits palatogenesis and arrests tooth development in a mouse model of the nevoid basal cell carcinoma syndrome. Dev Biol 331: 38–49.
16.
Cuervo, R., L. Covarrubias (2004) Death is the major fate of medial edge epithelial cells and the cause of basal lamina degradation during palatogenesis. Development 131: 15–24.
17.
Cui, X.M., Y. Chai, J. Chen, T. Yamamoto, Y. Ito, P. Bringas, C.F. Shuler (2003) TGF-beta3-dependent SMAD2 phosphorylation and inhibition of MEE proliferation during palatal fusion. Dev Dyn 227: 387–394.
18.
Dixon, M.J., J. Garner, M.W. Ferguson (1991) Immunolocalization of epidermal growth factor (EGF), EGF receptor and transforming growth factor alpha (TGF alpha) during murine palatogenesis in vivo and in vitro. Anat Embryol (Berl) 184: 83–91.
19.
Dudas, M., A. Nagy, N.J. Laping, A. Moustakas, V. Kaartinen (2004) TGF-beta3-induced palatal fusion is mediated by Alk-5/Smad pathway. Dev Biol 266: 96–108.
20.
Ferguson, M.W. (1988) Palate development. Development 103(suppl): 41–60.
21.
Fitchett, J.E., E.D. Hay (1989) Medial edge epithelium transforms to mesenchyme after embryonic palatal shelves fuse. Dev Biol 131: 455–474.
22.
Fitzpatrick, D.R., F. Denhez, P. Kondaiah, R.J. Akhurst (1990) Differential expression of TGF beta isoforms in murine palatogenesis. Development 109: 585–595.
23.
Gato, A., M.L. Martinez, C. Tudela, I. Alonso, J.A. Moro, M.A. Formoso, M.W. Ferguson, C. Martinez-Alvarez (2002) TGF-beta(3)-induced chondroitin sulphate proteoglycan mediates palatal shelf adhesion. Dev Biol 250: 393–405.
24.
Gawel-Thompson, K.J., R.M. Greene (1989) Epidermal growth factor: modulator of murine embryonic palate mesenchymal cell proliferation, polyamine biosynthesis, and polyamine transport. J Cell Physiol 140: 359–370.
25.
Gehris, A.L., M.M. Pisano, P. Nugent, R.M. Greene (1994) Regulation of TGF beta 3 gene expression in embryonic palatal tissue. In Vitro Cell Dev Biol Anim 30A: 671–679.
26.
Greene, R.M., R.M. Pratt (1976) Developmental aspects of secondary palate formation. J Embryol Exp Morphol 36: 225–245.
27.
Heldin, C.H., B. Westermark (1990) Signal transduction by the receptors for platelet-derived growth factor. J Cell Sci 96: 193–196.
28.
Hilliard, S.A., L. Yu, S. Gu, Z. Zhang, Y.P. Chen (2005) Regional regulation of palatal growth and patterning along the anterior-posterior axis in mice. J Anat 207: 655–667.
29.
Hudson, C.D., B.L. Shapiro (1973) A radioautographic study of deoxyribonucleic acid synthesis in embryonic rat palatal shelf epithelium with reference to the concept of programmed cell death. Arch Oral Biol 18: 77–84.
30.
Iamaroon, A., B. Tait, V.M. Diewert (1996) Cell proliferation and expression of EGF, TGF-alpha, and EGF receptor in the developing primary palate. J Dent Res 75: 1534–1539.
31.
Izvolsky, K.I., D. Shoykhet, Y. Yang, Q. Yu, M.A. Nugent, W.V. Cardoso (2003) Heparan sulfate-FGF10 interactions during lung morphogenesis. Dev Biol 258: 185–200.
32.
Jaskoll, T., H.A. Choy, H. Chen, M. Melnick (1996) Developmental expression and CORT-regulation of TGF-beta and EGF receptor mRNA during mouse palatal morphogenesis: correlation between CORT-induced cleft palate and TGF-beta 2 mRNA expression. Teratology 54: 34–44.
33.
Jin, J.Z., J. Ding (2006) Analysis of cell migration, transdifferentiation and apoptosis during mouse secondary palate fusion. Development 133: 3341–3347.
34.
Kaartinen, V., X.M. Cui, N. Heisterkamp, J. Groffen, C.F. Shuler (1997) Transforming growth factor-beta3 regulates transdifferentiation of medial edge epithelium during palatal fusion and associated degradation of the basement membrane. Dev Dyn 209: 255–260.
35.
Kaartinen, V., J.W. Voncken, C. Shuler, D. Warburton, D. Bu, N. Heisterkamp, J. Groffen (1995) Abnormal lung development and cleft palate in mice lacking TGF-beta 3 indicates defects of epithelial-mesenchymal interaction. Nat Genet 11: 415–421.
36.
Knight, A.S., B.C. Schutte, R. Jiang, M.J. Dixon (2006) Developmental expression analysis of the mouse and chick orthologues of IRF6: the gene mutated in Van der Woude syndrome. Dev Dyn 235: 1441–1447.
37.
Levi, G., S. Mantero, O. Barbieri, D. Cantatore, L. Paleari, A. Beverdam, F. Genova, B. Robert, G.R. Merlo (2006) Msx1 and Dlx5 act independently in development of craniofacial skeleton, but converge on the regulation of Bmp signaling in palate formation. Mech Dev 123: 3–16.
38.
Liu, W., X. Sun, A. Braut, Y. Mishina, R.R. Behringer, M. Mina, J.F. Martin (2005) Distinct functions for Bmp signaling in lip and palate fusion in mice. Development 132: 1453–1461.
39.
Lu, H., Y. Jin, G.L. Tipoe (2000) Alteration in the expression of bone morphogenetic protein-2,3,4,5 mRNA during pathogenesis of cleft palate in BALB/c mice. Arch Oral Biol 45: 133–140.
40.
Martinez-Alvarez, C., M.J. Blanco, R. Perez, M.A. Rabadan, M. Aparicio, E. Resel, T. Martinez, M.A. Nieto (2004) Snail family members and cell survival in physiological and pathological cleft palates. Dev Biol 265: 207–218.
41.
Martinez-Alvarez, C., R. Bonelli, C. Tudela, A. Gato, J. Mena, S. O’Kane, M.W. Ferguson (2000a) Bulging medial edge epithelial cells and palatal fusion. Int J Dev Biol 44: 331–335.
42.
Martinez-Alvarez, C., C. Tudela, J. Perez-Miguelsanz, S. O’Kane, J. Puerta, M.W. Ferguson (2000b) Medial edge epithelial cell fate during palatal fusion. Dev Biol 220: 343–357.
43.
Martinez-Sanz, E., A. Del Rio, C. Barrio, J. Murillo, E. Maldonado, B. Garcillan, M. Amoros, T. Fuerte, A. Fernandez, E. Trinidad M.A., Rabadan, Y. Lopez, M.L. Martinez, C. Martinez-Alvarez (2008) Alteration of medial-edge epithelium cell adhesion in two Tgf-beta3 null mouse strains. Differentiation 76: 417–430.
44.
Meng, L., Z. Bian, R. Torensma, J.W. Von den Hoff (2009) Biological mechanisms in palatogenesis and cleft palate. J Dent Res 88: 22–33.
45.
Miettinen, P.J., J.R. Chin, L. Shum, H.C. Slavkin, C.F. Shuler, R. Derynck, Z. Werb (1999) Epidermal growth factor receptor function is necessary for normal craniofacial development and palate closure. Nat Genet 22: 69–73.
46.
Mori, C., N. Nakamura, Y. Okamoto, M. Osawa, K. Shiota (1994) Cytochemical identification of programmed cell death in the fusing fetal mouse palate by specific labelling of DNA fragmentation. Anat Embryol (Berl) 190: 21–28.
47.
Murillo, J., E. Maldonado, M.C. Barrio, A. Del Rio, Y. Lopez, E. Martinez-Sanz, I. Gonzalez, C. Martin, I. Casado, C. Martinez-Alvarez (2009) Interactions between TGF-beta1 and TGF-beta3 and their role in medial edge epithelium cell death and palatal fusion in vitro. Differentiation 77: 209–220.
48.
Nawshad, A., D. Medici, C.C. Liu, E.D. Hay (2007) TGFbeta3 inhibits E-cadherin gene expression in palate medial-edge epithelial cells through a Smad2-Smad4-LEF1 transcription complex. J Cell Sci 120: 1646–1653.
49.
Nie, X.G. (2005) Differential expression of Bmp2, Bmp4 and Bmp3 in embryonic development of mouse anterior and posterior palate. Chin Med J (Engl) 118: 1710–1716.
50.
Nugent, P., R.M. Greene (1998) MSX-1 gene expression and regulation in embryonic palatal tissue. In Vitro Cell Dev Biol Anim 34: 831–835.
51.
Nugent, P., L. Ma, R.M. Greene (1998) Differential expression and biological activity of retinoic acid-induced TGFbeta isoforms in embryonic palate mesenchymal cells. J Cell Physiol 177: 36–46.
52.
Pantalacci, S., J. Prochazka, A. Martin, M. Rothova, A. Lambert, L. Bernard, C. Charles, L. Viriot, R. Peterkova , V. Laudet (2008) Patterning of palatal rugae through sequential addition reveals an anterior/posterior boundary in palatal development. BMC Dev Biol 8: 116.
53.
Patel, D., A. Lahiji, S. Patel, M. Franklin, X. Jimenez, D.J. Hicklin, X. Kang (2007) Monoclonal antibody cetuximab binds to and down-regulates constitutively activated epidermal growth factor receptor vIII on the cell surface. Anticancer Res 27: 3355–3366.
54.
Pelton, R.W., B.L. Hogan, D.A. Miller, H.L. Moses (1990) Differential expression of genes encoding TGFs beta1, beta2, and beta3 during murine palate formation. Dev Biol 141: 456–460.
55.
Pratt, R.M., G.R. Martin (1975) Epithelial cell death and cyclic AMP increase during palatal development. Proc Natl Acad Sci USA 72: 874–877.
56.
Proetzel, G., S.A. Pawlowski, M.V. Wiles, M. Yin, G.P. Boivin, P.N. Howles, J. Ding, M.W. Ferguson, T. Doetschman (1995) Transforming growth factor-beta 3 is required for secondary palate fusion. Nat Genet 11: 409–414.
57.
Qiu, C.X., M.W. Ferguson (1995) The distribution of PDGFs and PDGF-receptors during murine secondary palate development. J Anat 186: 17–29.
58.
Resel, E., E. Martínez-Sanz, I. González, E. Trinidad, B. Garcillán, M. Amorós, C. Alonso-Bañuelos, B. González-Meli, E. Lagarón, J. Murillo, A. Del Río, C. Barrio, M. López, C. Martínez-Álvarez (2007) In vitro manipulation of cleft palate connective tissue: setting the bases of a proposed new treatment. J Surg Res 138: 111–120.
59.
Rice, R., B. Spencer-Dene, E.C. Connor, A. Gritli-Linde, A.P. McMahon, C. Dickson, I. Thesleff, D.P. Rice (2004) Disruption of Fgf10/Fgfr2b-coordinated epithelial-mesenchymal interactions causes cleft palate. J Clin Invest 113: 1692–1700.
60.
Sasaki, Y., S. O’Kane, J. Dixon, M.J. Dixon, M.W. Ferguson (2007) Temporal and spatial expression of Pax9 and Sonic hedgehog during development of normal mouse palates and cleft palates in TGF-beta3 null embryos. Arch Oral Biol 52: 260–267.
61.
Satokata, I., R. Maas (1994) Msx1 deficient mice exhibit cleft palate and abnormalities of craniofacial and tooth development. Nat Genet 6: 348–356.
62.
Shah, R.M., A.V. Young, B.Z. Song, D.T. Wong (1994) A novel approach to the growth analysis of hamster secondary palate by histone 3 mRNA in situ hybridization. Histol Histopathol 9: 669–675.
63.
Sharpe, P.M., D.M. Foreman, M.J. Carette, S.L. Schor, M.W. Ferguson (1992) The effects of transforming growth factor-beta 1 on protein production by mouse embryonic palate mesenchymal cells in the presence or absence of serum. Arch Oral Biol 37: 39–48.
64.
Shiota, K., S. Fujita, T. Akiyama, C. Mori (1990) Expression of the epidermal growth factor receptor in developing fetal mouse palates: an immunohistochemical study. Am J Anat 188: 401–408.
65.
Shuler, C.F., D.E. Halpern, Y. Guo, A.C. Sank (1992) Medial edge epithelium fate traced by cell lineage analysis during epithelial-mesenchymal transformation in vivo. Dev Biol 154: 318–330.
66.
Takigawa, T., K. Shiota (2004) Terminal differentiation of palatal medial edge epithelial cells in vitro is not necessarily dependent on palatal shelf contact and midline epithelial seam formation. Int J Dev Biol 48: 307–317.
67.
Taya, Y., S. O’Kane, M.W. Ferguson (1999) Pathogenesis of cleft palate in TGF-beta3 knockout mice. Development 126: 3869–3879.
68.
Tudela, C., M.A. Formoso, T. Martinez, R. Perez, M. Aparicio, C. Maestro, A. Del Rio, E. Martinez, M. Ferguson, C. Martinez-Alvarez (2002) TGF-beta3 is required for the adhesion and intercalation of medial edge epithelial cells during palate fusion. Int J Dev Biol 46: 333–336.
69.
Turecková, J., C. Sahlberg, T. Aberg, J.V. Ruch, I. Thesleff, R. Peterkova (1995) Comparison of expression of the msx-1, msx-2, BMP-2 and BMP-4 genes in the mouse upper diastemal and molar tooth primordia. Int J Dev Biol 39: 459–468.
70.
Vaziri Sani, F., K. Hallberg, B.D. Harfe, A.P. McMahon, A. Linde, A. Gritli-Linde (2005) Fate-mapping of the epithelial seam during palatal fusion rules out epithelial-mesenchymal transformation. Dev Biol 285: 490–495.
71.
Weston, W.M., M.B. Potchinsky, C.M. Lafferty, L. Ma, R.M. Greene (1998) Cross-talk between signaling pathways in murine embryonic palate cells: effect of TGF beta and cAMP on EGF-induced DNA synthesis. In Vitro Cell Dev Biol Anim 34: 74–78.
72.
Xu, X., J. Han, Y. Ito, P. Bringas Jr., M.M. Urata, Y. Chai (2006) Cell autonomous requirement for Tgfbr2 in the disappearance of medial edge epithelium during palatal fusion. Dev Biol 297: 238–248.
73.
Yamamoto, T., X.M. Cui, C.F. Shuler (2003) Role of ERK1/2 signaling during EGF-induced inhibition of palatal fusion. Dev Biol 260: 512–521.
74.
Yang, L.T., V. Kaartinen (2007) Tgfb1 expressed in the Tgfb3 locus partially rescues the cleft palate phenotype of Tgfb3 null mutants. Dev Biol 312: 384–395.
75.
Yu, Z., J. Lin, Y. Xiao, J. Han, X. Zhang, H. Jia, Y. Tang , Y. Li (2005) Induction of cell-cycle arrest by all-trans retinoic acid in mouse embryonic palatal mesenchymal (MEPM) cells. Toxicol Sci 83: 349–354.
76.
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.
© 2010 S. Karger AG, Basel
2010
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.