Dynamic activation of Wnt, Fgf, and Hh signaling during soft palate development

Autoři: Eva Janečková aff001;  Jifan Feng aff001;  Jingyuan Li aff001;  Gabriela Rodriguez aff001;  Yang Chai aff001
Působiště autorů: Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, California, United States of America aff001
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: 10.1371/journal.pone.0223879


The soft palate is a key component of the oropharyngeal complex that is critical for swallowing, breathing, hearing and speech. However, complete functional restoration in patients with cleft soft palate remains a challenging task. New insights into the molecular signaling network governing the development of soft palate will help to overcome these clinical challenges. In this study, we investigated whether key signaling pathways required for hard palate development are also involved in soft palate development in mice. We described the dynamic expression patterns of signaling molecules from well-known pathways, such as Wnt, Hh, and Fgf, during the development of the soft palate. We found that Wnt signaling is active throughout the development of soft palate myogenic sites, predominantly in cells of cranial neural crest (CNC) origin neighboring the myogenic cells, suggesting that Wnt signaling may play a significant role in CNC-myogenic cell-cell communication during myogenic differentiation in the soft palate. Hh signaling is abundantly active in early palatal epithelium, some myogenic cells, and the CNC-derived cells adjacent to the myogenic cells. Hh signaling gradually diminishes during the later stages of soft palate development, indicating its involvement mainly in early embryonic soft palate development. Fgf signaling is expressed most prominently in CNC-derived cells in the myogenic sites and persists until later stages of embryonic soft palate development. Collectively, our results highlight a network of Wnt, Hh, and Fgf signaling that may be involved in the development of the soft palate, particularly soft palate myogenesis. These findings provide a foundation for future studies on the functional significance of these signaling pathways individually and collectively in regulating soft palate development.

Klíčová slova:

Epithelium – Hedgehog signaling – Muscle differentiation – Myosins – Wnt signaling cascade – Palate – Developmental signaling – Major histocompatibility complex


1. Lieberman DE. The evolution of the human head. 1st ed. Cambridge (MA): Belknap Press of Harvard University Press; 2011.

2. Tarr JT, Lambi AG, Bradley JP, Barbe MF, Popoff SN. Development of normal and cleft palate: A central role for connective tissue growth factor (CTGF)/CCN2. J Dev Biol. 2018;6(3):23.

3. Danescu A, Mattson M, Dool C, Diewert VM, Richman JM. Analysis of human soft palate morphogenesis supports regional regulation of palatal fusion. J Anat. 2015;227(4):474–86. doi: 10.1111/joa.12365 26299693

4. Chai Y, Maxson RE. Recent advances in craniofacial morphogenesis. Dev Dyn. 2006;235(9):2353–75. doi: 10.1002/dvdy.20833 16680722

5. Evans A, Ackermann B, Driscoll T. Functional anatomy of the soft palate applied to wind playing. Med Probl Perform Art. 2010;25(4):183–9. 21170481

6. Wehby GL, Cassell CH. The impact of orofacial clefts on quality of life and healthcare use and costs. Oral Dis. 2010;16(1):3–10. doi: 10.1111/j.1601-0825.2009.01588.x 19656316

7. Hunt O, Burden D, Hepper P, Johnston C. The psychosocial effects of cleft lip and palate: a systematic review. Eur J Orthod. 2005;27(3):274–85. doi: 10.1093/ejo/cji004 15947228

8. Monroy PLC, Grefte S, Kuijpers-Jagtman AM, Wagener FADTG, Von den Hoff JW. Strategies to improve regeneration of the soft palate muscles after cleft palate repair. Tissue Eng Part B Rev. 2012;18(6):468–77. doi: 10.1089/ten.TEB.2012.0049 22697475

9. Li J, Rodriguez G, Han X, Janeckova E, Kahng S, Song B, et al. Regulatory mechanisms of soft palate development and malformations. J Dent Res. 2019;98(9):959–67. doi: 10.1177/0022034519851786 31150594

10. Grimaldi A, Parada C, Chai Y. A comprehensive study of soft palate development in mice. Plos One. 2015;10(12):15.

11. Cordero DR, Brugmann S, Chu YN, Bajpai R, Jame M, Helms JA. Cranial neural crest cells on the move: their roles in craniofacial development. Am J Med Genet A. 2011;155A(2):270–9. doi: 10.1002/ajmg.a.33702 21271641

12. Iwata J, Suzuki A, Yokota T, Ho TV, Pelikan R, Urata M, et al. TGFβ regulates epithelial-mesenchymal interactions through WNT signaling activity to control muscle development in the soft palate. Development. 2014;141(4):909–17. doi: 10.1242/dev.103093 24496627

13. Sugii H, Grimaldi A, Li JY, Parada C, Thach VH, Feng JF, et al. The Dlx5-FGF10 signaling cascade controls cranial neural crest and myoblast interaction during oropharyngeal patterning and development. Development. 2017;144(21):4037–45. doi: 10.1242/dev.155176 28982687

14. Lin CX, Fisher AV, Yin Y, Maruyama T, Veith GM, Dhandha M, et al. The inductive role of Wnt-β-Catenin signaling in the formation of oral apparatus. Dev Biol. 2011;356(1):40–50. doi: 10.1016/j.ydbio.2011.05.002 21600200

15. Huelsken J, Vogel R, Brinkmann V, Erdmann B, Birchmeier C, Birchmeier W. Requirement for β-catenin in anterior-posterior axis formation in mice. J Cell Biol. 2000;148(3):567–78. doi: 10.1083/jcb.148.3.567 10662781

16. Brault V, Moore R, Kutsch S, Ishibashi M, Rowitch DH, McMahon AP, et al. Inactivation of the (β)-catenin gene by Wnt1-Cre-mediated deletion results in dramatic brain malformation and failure of craniofacial development. Development. 2001;128(8):1253–64. 11262227

17. Reid BS, Yang H, Melvin VS, Taketo MM, Williams T. Ectodermal Wnt/β-catenin signaling shapes the mouse face. Dev Biol. 2011;349(2):261–9. doi: 10.1016/j.ydbio.2010.11.012 21087601

18. Wang YP, Song LY, Zhou CJJ. The canonical Wnt/β-catenin signaling pathway regulates Fgf signaling for early facial development. Dev Biol. 2011;349(2):250–60. doi: 10.1016/j.ydbio.2010.11.004 21070765

19. Chen JQ, Lan Y, Baek JA, Gao Y, Jiang RL. Wnt/beta-catenin signaling plays an essential role in activation of odontogenic mesenchyme during early tooth development. Dev Biol. 2009;334(1):174–85. doi: 10.1016/j.ydbio.2009.07.015 19631205

20. Chiquet BT, Blanton SH, Burt A, Ma D, Stal S, Mulliken JB, et al. Variation in WNT genes is associated with non-syndromic cleft lip with or without cleft palate. Hum Mol Genet. 2008;17(14):2212–8. doi: 10.1093/hmg/ddn121 18413325

21. Fontoura C, Silva RM, Granjeiro JM, Letra A. Association of WNT9B gene polymorphisms with nonsyndromic cleft lip with or without cleft palate in Brazilian nuclear families. Cleft Palate Craniofac J. 2015;52(1):44–8. doi: 10.1597/13-146 24437584

22. Lan Y, Ryan RC, Zhang ZY, Bullard SA, Bush JO, Maltby KM, et al. Expression of Wnt9b and activation of canonical Wnt signaling during midfacial morphogenesis in mice. Dev Dyn. 2006;235(5):1448–54. doi: 10.1002/dvdy.20723 16496313

23. Suzuki A, Minamide R, Iwata J. WNT/β-catenin signaling plays a crucial role in myoblast fusion through regulation of nephrin expression during development. Development. 2018;145(23):8.

24. Zhong Z, Zhao H, Mayo J, Chai Y. Different requirements for Wnt signaling in tongue myogenic subpopulations. J Dent Res. 2015;94(3):421–9. doi: 10.1177/0022034514566030 25576472

25. Hutcheson DA, Zhao J, Merrell A, Haldar M, Kardon G. Embryonic and fetal limb myogenic cells are derived from developmentally distinct progenitors and have different requirements for β-catenin. Genes Dev. 2009;23(8):997–1013. doi: 10.1101/gad.1769009 19346403

26. Zhang XM, Ramalho-Santos M, McMahon AP. Smoothened mutants reveal redundant roles for Shh and Ihh signaling including regulation of L/R symmetry by the mouse node. Cell. 2001;106(2):781–92. 11517919

27. Jeong JH, Mao JH, Tenzen T, Kottmann AH, McMahon AP. Hedgehog signaling in the neural crest cells regulates the patterning and growth of facial primordia. Genes Dev. 2004;18(8):937–51. doi: 10.1101/gad.1190304 15107405

28. Lan Y, Jiang R. Sonic hedgehog signaling regulates reciprocal epithelial-mesenchymal interactions controlling palatal outgrowth. Development. 2009;136(8):1387–96. doi: 10.1242/dev.028167 19304890

29. Hammond NL, Brookes KJ, Dixon MJ. Ectopic Hedgehog signaling causes cleft palate and defective osteogenesis. J Dent Res. 2018;97(13):1485–93. doi: 10.1177/0022034518785336 29975848

30. Han J, Mayo J, Xu X, Li JY, Bringas P, Maas RL, et al. Indirect modulation of Shh signaling by Dlx5 affects the oral-nasal patterning of palate and rescues cleft palate in Msx1-null mice. Development. 2009;136(24):4225–33. doi: 10.1242/dev.036723 19934017

31. Rice R, Spencer-Dene B, Connor EC, Gritli-Linde A, McMahon AP, Dickson C, et al. Disruption of Fgf10/Fgfr2b-coordinated epithelial-mesenchymal interactions causes cleft palate. J Clin Invest. 2004;113(12):1692–700. doi: 10.1172/JCI20384 15199404

32. Sagai T, Amano T, Tamura M, Mizushina Y, Sumiyama K, Shiroishi T. A cluster of three long-range enhancers directs regional Shh expression in the epithelial linings. Development. 2009;136(10):1665–74. doi: 10.1242/dev.032714 19369396

33. Rice R, Connor E, Rice DPC. Expression patterns of Hedgehog signalling pathway members during mouse palate development. Gene Expr Patterns. 2006;6(2):206–12. doi: 10.1016/j.modgep.2005.06.005 16168717

34. Riley BM, Mansilla MA, Ma J, Daack-Hirsch S, Maher BS, Raffensperger LM, et al. Impaired FGF signaling contributes to cleft lip and palate. Proc Nat Acad Sci U.S.A. 2007;104(11):4512–7.

35. De Moerlooze L, Spencer-Dene B, Revest JM, Hajihosseini M, Rosewell I, Dickson C. An important role for the IIIb isoform of fibroblast growth factor receptor 2 (FGFR2) in mesenchymal-epithelial signalling during mouse organogenesis. Development. 2000;127(3):483–92. 10631169

36. Wang C, Chang JYF, Yang CF, Huang YQ, Liu JC, You P, et al. Type 1 Fibroblast growth factor receptor in cranial neural crest cell-derived mesenchyme is required for palatogenesis. J Biol Chem. 2013;288(30):22174–83. doi: 10.1074/jbc.M113.463620 23754280

37. Yu K, Karuppaiah K, Ornitz DM. Mesenchymal fibroblast growth factor receptor signaling regulates palatal shelf elevation during secondary palate formation. Dev Dyn. 2015;244(11):1427–38. doi: 10.1002/dvdy.24319 26250517

38. Jin JZ, Lei ZM, Lan ZJ, Mukhopadhyay P, Ding JX. Inactivation of Fgfr2 gene in mouse secondary palate mesenchymal cells leads to cleft palate. Reprod Toxicol. 2018;77:137–42. doi: 10.1016/j.reprotox.2018.03.004 29526646

39. Weng MJ, Chen ZX, Xiao Q, Li RM, Chen ZQ. A review of FGF signaling in palate development. Biomed Pharmacother. 2018;103:240–7. doi: 10.1016/j.biopha.2018.04.026 29655165

40. Olwin BB, Arthur K, Hannon K, Hein P, McFall A, Riley B, et al. Role of Fgfs in skeletal muscle and limb development. Mol Reprod Dev. 1994;39(1):90–101. doi: 10.1002/mrd.1080390114 7999366

41. Ciruna B, Rossant J. Fgf signaling regulates mesoderm cell fate specification and morphogenetic movement at the primitive streak. Dev Cell. 2001;1(1):37–49. 11703922

42. Smith TM, Lozanoff S, Iyyanar PP, Nazarali AJ. Molecular signaling along the anterior-posterior axis of early palate development. Front Physiol. 2013;3:14.

43. Reynolds K, Kumari P, Rincon LS, Gu R, Ji Y, Kumar S, et al. Wnt signaling in orofacial clefts: crosstalk, pathogenesis and models. Dis Models Mech. 2019;12(2):24.

44. Lee JM, Kim JY, Cho KW, Lee MJ, Cho SW, Kwak S, et al. Wnt11/Fgfr1b cross-talk modulates the fate of cells in palate development. Dev Biol. 2008;314(2):341–50. doi: 10.1016/j.ydbio.2007.11.033 18191119

45. Bai CB, Auerbach W, Lee JS, Stephen D, Joyner AL. Gli2, but not Gli1, is required for initial Shh signaling and ectopic activation of the Shh pathway. Development. 2002;129(20):4753–61. 12361967

46. Lustig B, Jerchow B, Sachs M, Weiler S, Pietsch T, Karsten U, et al. Negative feedback loop of Wnt signaling through upregulation of conductin/Axin2 in colorectal and liver tumors. Mol Cell Biol. 2002;22(4):1184–93. doi: 10.1128/MCB.22.4.1184-1193.2002 11809809

47. Menezes R, Letra A, Kim AH, Kuchler EC, Day A, Tannure PN, et al. Studies with Wnt genes and nonsyndromic cleft lip and palate. Birth Defects Res A Clin Mol Teratol. 2010;88(11):995–1000. doi: 10.1002/bdra.20720 20890934

48. He FL, Xiong W, Wang Y, Li L, Liu C, Yamagami T, et al. Epithelial Wnt/β-catenin signaling regulates palatal shelf fusion through regulation of Tgfβ3 expression. Dev Biol. 2011;350(2):511–9. doi: 10.1016/j.ydbio.2010.12.021 21185284

49. Hagemann AIH, Scholpp S. The tale of the three brothers—Shh, Wnt, and Fgf during development of the thalamus. Front Neurosci. 2012;6:9.

50. Yang YZ, Kozin SH. Cell signaling regulation of vertebrate limb growth and patterning. J Bone Joint Surg Am. 2009;91A:76–80.

51. Aman AJ, Fulbright AN, Parichy DM. Wnt/β-catenin regulates an ancient signaling network during zebrafish scale development. Elife. 2018;7:22.

52. Tucker A, Sharpe P. The cutting-edge of mammalian development; How the embryo makes teeth. Nat Rev Genet. 2004;5(7):499–508. doi: 10.1038/nrg1380 15211352

53. Liu F, Wang SL. Molecular cues for development and regeneration of salivary glands. Histol Histopathol. 2014;29(3):305–12. doi: 10.14670/HH-29.305 24189993

54. Tucker AS. Salivary gland development. Semin Cell Dev Biol. 2007;18(2):237–44 doi: 10.1016/j.semcdb.2007.01.006 17336109

55. Volckaert T, De Langhe SP. Wnt and FGF mediated epithelial-mesenchymal crosstalk during lung development. Dev Dyn. 2015;244(3):342–66. doi: 10.1002/dvdy.24234 25470458

56. Warner DR, Smith HS, Webb CL, Greene RM, Pisano MM. Expression of Wnts in the developing murine secondary palate. Int J Dev Biol. 2009;53(7):1105–12. doi: 10.1387/ijdb.082578dw 19598129

57. Jin YR, Han XH, Taketo MM, Yoon JK. Wnt9b-dependent FGF signaling is crucial for outgrowth of the nasal and maxillary processes during upper jaw and lip development. Development. 2012;139(10):1821–30. doi: 10.1242/dev.075796 22461561

58. Alappat SR, Zhang ZY, Suzuki K, Zhang XY, Liu HB, Jiang RL, et al. The cellular and molecular etiology of the cleft secondary palate in Fgf10 mutant mice. Dev Biol. 2005;277(1):102–13. doi: 10.1016/j.ydbio.2004.09.010 15572143

59. Teshima THN, Lourenco SV, Tucker AS. Multiple cranial organ defects after conditionally knocking out Fgf10 in the neural crest. Front Physiol. 2016;7:10.

60. Puthiyaveetil JSV, Kota K, Chakkarayan R, Chakkarayan J, Thodiyil AKP. Epithelial—mesenchymal interactions in tooth development and the significant role of growth factors and genes with emphasis on mesenchyme—A review. J Clin Diagn Res. 2016;10(9):ZE5–ZE9.

61. Ribatti D, Santoiemma M. Epithelial-mesenchymal interactions: a fundamental developmental biology mechanism. Int J Dev Biol. 2014;58(5):303–6. doi: 10.1387/ijdb.140143dr 25354449

62. Cunha GR, Hom YK. Role of Mesenchymal-epithelial interactions in mammary gland development. J Mammary Gland Biol Neoplasia. 1996;1(1):21–35. 10887478

63. Iwata J, Parada C, Chai Y. The mechanism of TGF-β signaling during palate development. Oral Dis. 2011;17(8):733–44.

64. Deb A. Cell cell interaction in the heart via Wnt/β-catenin pathway after cardiac injury. Cardiovasc Res. 2014;102(2):214–23. doi: 10.1093/cvr/cvu054 24591151

65. Hosokawa R, Oka K, Yamaza T, Iwata J, Urata M, Xu X, et al. TGF-beta mediated Fgf10 signaling in cranial neural crest cells controls development of myogenic progenitor cells through tissue-tissue interactions during tongue morphogenesis. Dev Biol. 2010;341(1):186–95. doi: 10.1016/j.ydbio.2010.02.030 20193675

Článek vyšel v časopise


2019 Číslo 10