Cleft palate is among the most common birth defects in humans. Previous studies have shown that Shh signaling plays critical roles in palate development and regulates expression of several members of the forkhead-box (Fox) family transcription factors, including Foxf1 and Foxf2, in the facial primordia. Although cleft palate has been reported in mice deficient in Foxf2, whether Foxf2 plays an intrinsic role in and how Foxf2 regulates palate development remain to be elucidated. Using Cre/loxP-mediated tissue-specific gene inactivation in mice, we show that Foxf2 is required in the neural crest-derived palatal mesenchyme for normal palatogenesis. We found that Foxf2 mutant embryos exhibit altered patterns of expression of Shh, Ptch1, and Shox2 in the developing palatal shelves. Through RNA-seq analysis, we identified over 150 genes whose expression was significantly up- or down-regulated in the palatal mesenchyme in Foxf2-/- mutant embryos in comparison with control littermates. Whole mount in situ hybridization analysis revealed that the Foxf2 mutant embryos exhibit strikingly corresponding patterns of ectopic Fgf18 expression in the palatal mesenchyme and concomitant loss of Shh expression in the palatal epithelium in specific subdomains of the palatal shelves that correlate with where Foxf2, but not Foxf1, is expressed during normal palatogenesis. Furthermore, tissue specific inactivation of both Foxf1 and Foxf2 in the early neural crest cells resulted in ectopic activation of Fgf18 expression throughout the palatal mesenchyme and dramatic loss of Shh expression throughout the palatal epithelium. Addition of exogenous Fgf18 protein to cultured palatal explants inhibited Shh expression in the palatal epithelium. Together, these data reveal a novel Shh-Foxf-Fgf18-Shh circuit in the palate development molecular network, in which Foxf1 and Foxf2 regulate palatal shelf growth downstream of Shh signaling, at least in part, by repressing Fgf18 expression in the palatal mesenchyme to ensure maintenance of Shh expression in the palatal epithelium.
References
[1]
Bush JO, Jiang R. Palatogenesis: morphogenetic and molecular mechanisms of secondary palate development. Development. 2012;139(2):231–243. doi: 10.1242/dev.067082. pmid:22186724
Gritli-Linde A. Molecular control of secondary palate development. Developmental biology. 2007;301(2):309–326. pmid:16942766 doi: 10.1016/j.ydbio.2006.07.042
[4]
Chai Y, Maxson RE Jr. Recent advances in craniofacial morphogenesis. Developmental dynamics: an official publication of the American Association of Anatomists. 2006;235(9):2353–2375. doi: 10.1002/dvdy.20833
[5]
Rice R, Connor E, Rice DP. Expression patterns of Hedgehog signalling pathway members during mouse palate development. Gene expression patterns: GEP. 2006;6(2):206–212. pmid:16168717 doi: 10.1016/j.modgep.2005.06.005
[6]
Jiang J, Hui CC. Hedgehog signaling in development and cancer. Developmental cell. 2008;15(6):801–812. doi: 10.1016/j.devcel.2008.11.010. pmid:19081070
[7]
Zhang Z, Song Y, Zhao X, Zhang X, Fermin C, Chen Y. 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. 2002;129(17):4135–4146. pmid:12163415
[8]
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. The Journal of clinical investigation. 2004;113(12):1692–1700. pmid:15199404 doi: 10.1172/jci20384
[9]
Jeong J, Mao J, Tenzen T, Kottmann AH, McMahon AP. Hedgehog signaling in the neural crest cells regulates the patterning and growth of facial primordia. Genes & development. 2004;18(8):937–951. doi: 10.1101/gad.1190304
[10]
Lan Y, Jiang R. Sonic hedgehog signaling regulates reciprocal epithelial-mesenchymal interactions controlling palatal outgrowth. Development. 2009;136(8):1387–1396. doi: 10.1242/dev.028167. pmid:19304890
[11]
Li L, Lin M, Wang Y, Cserjesi P, Chen Z, Chen Y. BmprIa is required in mesenchymal tissue and has limited redundant function with BmprIb in tooth and palate development. Developmental biology. 2011;349(2):451–461. doi: 10.1016/j.ydbio.2010.10.023. pmid:21034733
[12]
Baek JA, Lan Y, Liu H, Maltby KM, Mishina Y, Jiang R. Bmpr1a signaling plays critical roles in palatal shelf growth and palatal bone formation. Developmental biology. 2011;350(2):520–531. doi: 10.1016/j.ydbio.2010.12.028. pmid:21185278
[13]
Kaufmann E, Knochel W. Five years on the wings of fork head. Mechanisms of development. 1996;57(1):3–20. pmid:8817449 doi: 10.1016/0925-4773(96)00539-4
[14]
Clark KL, Halay ED, Lai E, Burley SK. Co-crystal structure of the HNF-3/fork head DNA-recognition motif resembles histone H5. Nature. 1993;364(6436):412–420. pmid:8332212 doi: 10.1038/364412a0
[15]
Wang T, Tamakoshi T, Uezato T, Shu F, Kanzaki-Kato N, Fu Y, et al. Forkhead transcription factor Foxf2 (LUN)-deficient mice exhibit abnormal development of secondary palate. Developmental biology. 2003;259(1):83–94. pmid:12812790 doi: 10.1016/s0012-1606(03)00176-3
[16]
Hellqvist M, Mahlapuu M, Samuelsson L, Enerback S, Carlsson P. Differential activation of lung-specific genes by two forkhead proteins, FREAC-1 and FREAC-2. The Journal of biological chemistry. 1996;271(8):4482–4490. pmid:8626802 doi: 10.1074/jbc.271.8.4482
[17]
Hellqvist M, Mahlapuu M, Blixt A, Enerback S, Carlsson P. The human forkhead protein FREAC-2 contains two functionally redundant activation domains and interacts with TBP and TFIIB. The Journal of biological chemistry. 1998;273(36):23335–23343. pmid:9722567 doi: 10.1074/jbc.273.36.23335
[18]
Mahlapuu M, Pelto-Huikko M, Aitola M, Enerback S, Carlsson P. FREAC-1 contains a cell-type-specific transcriptional activation domain and is expressed in epithelial-mesenchymal interfaces. Developmental biology. 1998;202(2):183–195. pmid:9769171 doi: 10.1006/dbio.1998.9010
[19]
Mahlapuu M, Ormestad M, Enerback S, Carlsson P. The forkhead transcription factor Foxf1 is required for differentiation of extra-embryonic and lateral plate mesoderm. Development. 2001;128(2):155–166. pmid:11124112
[20]
Jochumsen U, Werner R, Miura N, Richter-Unruh A, Hiort O, Holterhus PM. Mutation analysis of FOXF2 in patients with disorders of sex development (DSD) in combination with cleft palate. Sexual development: genetics, molecular biology, evolution, endocrinology, embryology, and pathology of sex determination and differentiation. 2008;2(6):302–308. doi: 10.1159/000195679
[21]
Bolte C, Ren X, Tomley T, Ustiyan V, Pradhan A, Hoggatt A, et al. Forkhead box F2 regulation of platelet-derived growth factor and myocardin/serum response factor signaling is essential for intestinal development. The Journal of biological chemistry. 2015;290(12):7563–7575. doi: 10.1074/jbc.M114.609487. pmid:25631042
[22]
Danielian PS, Muccino D, Rowitch DH, Michael SK, McMahon AP. Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Current biology: CB. 1998;8(24):1323–1326. pmid:9843687 doi: 10.1016/s0960-9822(07)00562-3
[23]
Chai Y, Jiang X, Ito Y, Bringas P Jr., Han J Rowitch DH, et al. Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. Development. 2000;127(8):1671–1679. pmid:10725243
[24]
Han J, Ito Y, Yeo JY, Sucov HM, Maas R, Chai Y. Cranial neural crest-derived mesenchymal proliferation is regulated by Msx1-mediated p19(INK4d) expression during odontogenesis. Developmental biology. 2003;261(1):183–196. pmid:12941628 doi: 10.1016/s0012-1606(03)00300-2
[25]
Lan Y, Wang Q, Ovitt CE, Jiang R. A unique mouse strain expressing Cre recombinase for tissue-specific analysis of gene function in palate and kidney development. Genesis. 2007;45(10):618–624. pmid:17941042 doi: 10.1002/dvg.20334
[26]
Pantalacci S, Prochazka J, Martin A, Rothova M, Lambert A, Bernard L, et al. Patterning of palatal rugae through sequential addition reveals an anterior/posterior boundary in palatal development. BMC developmental biology. 2008;8:116. doi: 10.1186/1471-213X-8-116. pmid:19087265
[27]
Welsh IC, O'Brien TP. Signaling integration in the rugae growth zone directs sequential SHH signaling center formation during the rostral outgrowth of the palate. Developmental biology. 2009;336(1):53–67. doi: 10.1016/j.ydbio.2009.09.028. pmid:19782673
[28]
Han J, Mayo J, Xu X, Li J, Bringas P Jr., 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):4225–4233. doi: 10.1242/dev.036723. pmid:19934017
[29]
Zhou J, Gao Y, Lan Y, Jia S, Jiang R. Pax9 regulates a molecular network involving Bmp4, Fgf10, Shh signaling and the Osr2 transcription factor to control palate morphogenesis. Development. 2013;140(23):4709–4718. doi: 10.1242/dev.099028. pmid:24173808
[30]
Marigo V, Scott MP, Johnson RL, Goodrich LV, Tabin CJ. Conservation in hedgehog signaling: induction of a chicken patched homolog by Sonic hedgehog in the developing limb. Development. 1996;122(4):1225–1233. pmid:8620849
[31]
Marigo V, Tabin CJ. Regulation of patched by sonic hedgehog in the developing neural tube. Proceedings of the National Academy of Sciences of the United States of America. 1996;93(18):9346–9351. pmid:8790332 doi: 10.1073/pnas.93.18.9346
[32]
Goodrich LV, Johnson RL, Milenkovic L, McMahon JA, Scott MP. Conservation of the hedgehog/patched signaling pathway from flies to mice: induction of a mouse patched gene by Hedgehog. Genes & development. 1996;10(3):301–312. doi: 10.1101/gad.10.3.301
[33]
Alexandre C, Jacinto A, Ingham PW. Transcriptional activation of hedgehog target genes in Drosophila is mediated directly by the cubitus interruptus protein, a member of the GLI family of zinc finger DNA-binding proteins. Genes & development. 1996;10(16):2003–2013. doi: 10.1101/gad.10.16.2003
[34]
Yu L, Gu S, Alappat S, Song Y, Yan M, Zhang X, et al. Shox2-deficient mice exhibit a rare type of incomplete clefting of the secondary palate. Development. 2005;132(19):4397–4406. pmid:16141225 doi: 10.1242/dev.02013
[35]
Smith TM, Lozanoff S, Iyyanar PP, Nazarali AJ. Molecular signaling along the anterior-posterior axis of early palate development. Frontiers in physiology. 2012;3:488. doi: 10.3389/fphys.2012.00488. pmid:23316168
[36]
Lan Y, Kingsley PD, Cho ES, Jiang R. Osr2, a new mouse gene related to Drosophila odd-skipped, exhibits dynamic expression patterns during craniofacial, limb, and kidney development. Mechanisms of development. 2001;107(1–2):175–179. pmid:11520675 doi: 10.1016/s0925-4773(01)00457-9
[37]
Economou AD, Ohazama A, Porntaveetus T, Sharpe PT, Kondo S, Basson MA, et al. Periodic stripe formation by a Turing mechanism operating at growth zones in the mammalian palate. Nature genetics. 2012;44(3):348–351. doi: 10.1038/ng.1090. pmid:22344222
[38]
Cobourne MT, Green JB. Hedgehog signalling in development of the secondary palate. Frontiers of oral biology. 2012;16:52–59. doi: 10.1159/000337543. pmid:22759669
[39]
Mo R, Freer AM, Zinyk DL, Crackower MA, Michaud J, Heng HH, et al. Specific and redundant functions of Gli2 and Gli3 zinc finger genes in skeletal patterning and development. Development. 1997;124(1):113–123. pmid:9006072
[40]
Huang X, Goudy SL, Ketova T, Litingtung Y, Chiang C. Gli3-deficient mice exhibit cleft palate associated with abnormal tongue development. Developmental dynamics: an official publication of the American Association of Anatomists. 2008;237(10):3079–3087. doi: 10.1002/dvdy.21714
[41]
Hoffmann AD, Yang XH, Burnicka-Turek O, Bosman JD, Ren X, Steimle JD, et al. Foxf genes integrate tbx5 and hedgehog pathways in the second heart field for cardiac septation. PLoS genetics. 2014;10(10):e1004604. doi: 10.1371/journal.pgen.1004604. pmid:25356765
[42]
Ormestad M, Astorga J, Carlsson P. Differences in the embryonic expression patterns of mouse Foxf1 and -2 match their distinct mutant phenotypes. Developmental dynamics: an official publication of the American Association of Anatomists. 2004;229(2):328–333. doi: 10.1002/dvdy.10426
[43]
Ormestad M, Astorga J, Landgren H, Wang T, Johansson BR, Miura N, et al. Foxf1 and Foxf2 control murine gut development by limiting mesenchymal Wnt signaling and promoting extracellular matrix production. Development. 2006;133(5):833–843. pmid:16439479 doi: 10.1242/dev.02252
[44]
Mahlapuu M, Enerback S, Carlsson P. Haploinsufficiency of the forkhead gene Foxf1, a target for sonic hedgehog signaling, causes lung and foregut malformations. Development. 2001;128(12):2397–2406. pmid:11493558
[45]
Nie X, Luukko K, Kettunen P. FGF signalling in craniofacial development and developmental disorders. Oral diseases. 2006;12(2):102–111. pmid:16476029 doi: 10.1111/j.1601-0825.2005.01176.x
[46]
Belov AA, Mohammadi M. Molecular mechanisms of fibroblast growth factor signaling in physiology and pathology. Cold Spring Harbor perspectives in biology. 2013;5(6). doi: 10.1101/cshperspect.a015958
[47]
Belleudi F, Leone L, Nobili V, Raffa S, Francescangeli F, Maggio M, et al. Keratinocyte growth factor receptor ligands target the receptor to different intracellular pathways. Traffic. 2007;8(12):1854–1872. pmid:17944804 doi: 10.1111/j.1600-0854.2007.00651.x
[48]
Francavilla C, Rigbolt KT, Emdal KB, Carraro G, Vernet E, Bekker-Jensen DB, et al. Functional proteomics defines the molecular switch underlying FGF receptor trafficking and cellular outputs. Molecular cell. 2013;51(6):707–722. doi: 10.1016/j.molcel.2013.08.002. pmid:24011590
[49]
Olsen SK, Li JY, Bromleigh C, Eliseenkova AV, Ibrahimi OA, Lao Z, et al. Structural basis by which alternative splicing modulates the organizer activity of FGF8 in the brain. Genes & development. 2006;20(2):185–198. doi: 10.1101/gad.1365406
[50]
Brown A, Adam LE, Blundell TL. The crystal structure of fibroblast growth factor 18 (FGF18). Protein & cell. 2014;5(5):343–347. doi: 10.1007/s13238-014-0033-4
[51]
Davidson D, Blanc A, Filion D, Wang H, Plut P, Pfeffer G, et al. Fibroblast growth factor (FGF) 18 signals through FGF receptor 3 to promote chondrogenesis. The Journal of biological chemistry. 2005;280(21):20509–20515. pmid:15781473 doi: 10.1074/jbc.m410148200
[52]
Miyaoka Y, Tanaka M, Imamura T, Takada S, Miyajima A. A novel regulatory mechanism for Fgf18 signaling involving cysteine-rich FGF receptor (Cfr) and delta-like protein (Dlk). Development. 2010;137(1):159–167. doi: 10.1242/dev.041574. pmid:20023171
[53]
Ohbayashi N, Shibayama M, Kurotaki Y, Imanishi M, Fujimori T, Itoh N, et al. FGF18 is required for normal cell proliferation and differentiation during osteogenesis and chondrogenesis. Genes & development. 2002;16(7):870–879. doi: 10.1101/gad.965702
[54]
Liu Z, Xu J, Colvin JS, Ornitz DM. Coordination of chondrogenesis and osteogenesis by fibroblast growth factor 18. Genes & development. 2002;16(7):859–869. doi: 10.1101/gad.965602
[55]
Riley BM, Mansilla MA, Ma J, Daack-Hirsch S, Maher BS, Raffensperger LM, et al. Impaired FGF signaling contributes to cleft lip and palate. Proceedings of the National Academy of Sciences of the United States of America. 2007;104(11):4512–4517. pmid:17360555 doi: 10.1073/pnas.0607956104
[56]
Ren X, Ustiyan V, Pradhan A, Cai Y, Havrilak JA, Bolte CS, et al. FOXF1 transcription factor is required for formation of embryonic vasculature by regulating VEGF signaling in endothelial cells. Circulation research. 2014;115(8):709–720. doi: 10.1161/CIRCRESAHA.115.304382. pmid:25091710
[57]
Zhang Y, Zhao X, Hu Y, St Amand T, Zhang M, Ramamurthy R, et al. Msx1 is required for the induction of Patched by Sonic hedgehog in the mammalian tooth germ. Developmental dynamics: an official publication of the American Association of Anatomists. 1999;215(1):45–53. doi: 10.1002/(sici)1097-0177(199905)215:1<45::aid-dvdy5>3.3.co;2-x
[58]
Brunskill EW, Potter SS. RNA-Seq defines novel genes, RNA processing patterns and enhancer maps for the early stages of nephrogenesis: Hox supergenes. Developmental biology. 2012;368(1):4–17. doi: 10.1016/j.ydbio.2012.05.030. pmid:22664176
