Histochemistry and Cell Biology

, Volume 146, Issue 3, pp 289–300 | Cite as

Long-range enhancers modulate Foxf1 transcription in blood vessels of pulmonary vascular network

  • Hyejin Seo
  • Jinsun Kim
  • Gi-Hee Park
  • Yuri Kim
  • Sung-Won ChoEmail author
Original Paper


Intimate crosstalk occurs between the pulmonary epithelium and the vascular network during lung development. The transcription factor forkhead box f1 (Foxf1) is expressed in the lung mesenchyme and plays an indispensable role in pulmonary angiogenesis. Sonic hedgehog (Shh), a signalling molecule, is expressed in lung epithelium and is required to establish proper angiogenesis. It has been suggested that Foxf1, a downstream target of the Shh signalling pathway, mediates interaction between angiogenesis and the epithelium in lung. However, there has been no clear evidence showing the mechanism how Foxf1 is regulated by Shh signalling pathway during lung development. In this study, we investigated the lung-specific enhancers of Foxf1 and the Gli binding on the enhancers. At first, we found three evolutionarily conserved Foxf1 enhancers, two of which were long-range enhancers. Of the long-range enhancers, one demonstrated tissue-specific activity in the proximal and distal pulmonary blood vessels, while the other one demonstrated activity only in distal blood vessels. At analogous positions in human, these long-range enhancers were included in a regulatory region that was reportedly repeatedly deleted in alveolar capillary dysplasia with misalignment of pulmonary vein patients, which indicates the importance of these enhancers in pulmonary blood vessel formation. We also determined that Gli increased the activity of one of these long-range enhancers, which was specific to distal blood vessel, suggesting that Shh regulates Foxf1 transcription in pulmonary distal blood vessel formation.


Foxf1 Shh Enhancer Angiogenesis Lung development Endothelium 



We thank Prof. Shiroishi (National Institute of Genetics, Japan) for kindly providing pHSF51 plasmid. We thank H. K. Kim and J. K. Cho for animal care and J. W. Bok and M. H. Kim for constructive discussions and experimental guidance.

Authors’ contribution

H.S. and J.K. designed and performed the experiments and prepared the manuscript. Y.K. and G.H.P. helped with some experiments. S.W.C. coordinated the experimental design and prepared the manuscript.


This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MEST) (NRF-2012M3A9B2052522). This research was supported by BK21 PLUS Project, Yonsei University College of Dentistry.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

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  1. Astorga J, Carlsson P (2007) Hedgehog induction of murine vasculogenesis is mediated by Foxf1 and Bmp4. Development 134:3753–3761. doi: 10.1242/dev.004432 CrossRefPubMedGoogle Scholar
  2. Bellusci S, Furuta Y, Rush MG, Henderson R, Winnier G, Hogan BL (1997) Involvement of Sonic hedgehog (Shh) in mouse embryonic lung growth and morphogenesis. Development 124:53–63Google Scholar
  3. Cattelino A et al (2003) The conditional inactivation of the beta-catenin gene in endothelial cells causes a defective vascular pattern and increased vascular fragility. J Cell Biol 162:1111–1122. doi: 10.1083/jcb.200212157 CrossRefPubMedPubMedCentralGoogle Scholar
  4. Chang VW, Ho Y (2001) Structural characterization of the mouse Foxf1a gene. Gene 267:201–211CrossRefPubMedGoogle Scholar
  5. Dekker J, Rippe K, Dekker M, Kleckner N (2002) Capturing chromosome conformation. Science 295:1306–1311. doi: 10.1126/science.1067799 CrossRefPubMedGoogle Scholar
  6. Gebb SA, Shannon JM (2000) Tissue interactions mediate early events in pulmonary vasculogenesis. Dev Dyn 217:159–169. doi: 10.1002/(SICI)1097-0177(200002)217:2<159:AID-DVDY3>3.0.CO;2-9 CrossRefPubMedGoogle Scholar
  7. Grindley JC, Bellusci S, Perkins D, Hogan BL (1997) Evidence for the involvement of the Gli gene family in embryonic mouse lung development. Dev Biol 188:337–348. doi: 10.1006/dbio.1997.8644 CrossRefPubMedGoogle Scholar
  8. Healy AM, Morgenthau L, Zhu X, Farber HW, Cardoso WV (2000) VEGF is deposited in the subepithelial matrix at the leading edge of branching airways and stimulates neovascularization in the murine embryonic lung. Dev Dyn 219:341–352. doi: 10.1002/1097-0177(2000)9999:9999<:AID-DVDY1061>3.0.CO;2-M CrossRefPubMedGoogle Scholar
  9. Herriges MJ, Swarr DT, Morley MP, Rathi KS, Peng T, Stewart KM, Morrisey EE (2014) Long noncoding RNAs are spatially correlated with transcription factors and regulate lung development. Genes Dev 28:1363–1379. doi: 10.1101/gad.238782.114 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Hines EA, Sun X (2014) Tissue crosstalk in lung development. J Cell Biochem 115:1469–1477. doi: 10.1002/jcb.24811 CrossRefPubMedGoogle Scholar
  11. Kalinichenko VV, Lim L, Shin B, Costa RH (2001a) Differential expression of forkhead box transcription factors following butylated hydroxytoluene lung injury. Am J Physiol Lung Cell Mol Physiol 280:L695–L704PubMedGoogle Scholar
  12. Kalinichenko VV et al (2001b) Defects in pulmonary vasculature and perinatal lung hemorrhage in mice heterozygous null for the Forkhead Box f1 transcription factor. Dev Biol 235:489–506. doi: 10.1006/dbio.2001.0322 CrossRefPubMedGoogle Scholar
  13. Kalinichenko VV, Zhou Y, Bhattacharyya D, Kim W, Shin B, Bambal K, Costa RH (2002) Haploinsufficiency of the mouse Forkhead Box f1 gene causes defects in gall bladder development. J Biol Chem 277:12369–12374. doi: 10.1074/jbc.M112162200 CrossRefPubMedGoogle Scholar
  14. Keijzer R, van Tuyl M, Meijers C, Post M, Tibboel D, Grosveld F, Koutsourakis M (2001) The transcription factor GATA6 is essential for branching morphogenesis and epithelial cell differentiation during fetal pulmonary development. Development 128:503–511PubMedGoogle Scholar
  15. Kim IM, Zhou Y, Ramakrishna S, Hughes DE, Solway J, Costa RH, Kalinichenko VV (2005) Functional characterization of evolutionarily conserved DNA regions in forkhead box f1 gene locus. J Biol Chem 280:37908–37916. doi: 10.1074/jbc.M506531200 CrossRefPubMedGoogle Scholar
  16. Lin Q, Lu J, Yanagisawa H, Webb R, Lyons GE, Richardson JA, Olson EN (1998) Requirement of the MADS-box transcription factor MEF2C for vascular development. Development 125:4565–4574PubMedGoogle Scholar
  17. Litingtung Y, Lei L, Westphal H, Chiang C (1998) Sonic hedgehog is essential to foregut development. Nat Genet 20:58–61. doi: 10.1038/1717 CrossRefPubMedGoogle Scholar
  18. Madison BB, McKenna LB, Dolson D, Epstein DJ, Kaestner KH (2009) FoxF1 and FoxL1 link hedgehog signaling and the control of epithelial proliferation in the developing stomach and intestine. J Biol Chem 284:5936–5944. doi: 10.1074/jbc.M808103200 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Mahlapuu M, Enerback S, Carlsson P (2001a) Haploinsufficiency of the forkhead gene Foxf1, a target for sonic hedgehog signaling, causes lung and foregut malformations. Development 128:2397–2406PubMedGoogle Scholar
  20. Mahlapuu M, Ormestad M, Enerback S, Carlsson P (2001b) The forkhead transcription factor Foxf1 is required for differentiation of extra-embryonic and lateral plate mesoderm. Development 128:155–166PubMedGoogle Scholar
  21. McGrath KE, Koniski AD, Malik J, Palis J (2003) Circulation is established in a stepwise pattern in the mammalian embryo. Blood 101:1669–1676. doi: 10.1182/blood-2002-08-2531 CrossRefPubMedGoogle Scholar
  22. Melboucy-Belkhir S et al (2014) Forkhead Box F1 represses cell growth and inhibits COL1 and ARPC2 expression in lung fibroblasts in vitro. Am J Physiol Lung Cell Mol Physiol 307:L838–L847. doi: 10.1152/ajplung.00012.2014 CrossRefPubMedGoogle Scholar
  23. Miller LA, Wert SE, Clark JC, Xu Y, Perl AK, Whitsett JA (2004) Role of Sonic hedgehog in patterning of tracheal-bronchial cartilage and the peripheral lung. Dev Dyn 231:57–71. doi: 10.1002/dvdy.20105 CrossRefPubMedGoogle Scholar
  24. Motoyama J, Liu J, Mo R, Ding Q, Post M, Hui CC (1998) Essential function of Gli2 and Gli3 in the formation of lung, trachea and oesophagus. Nat Genet 20:54–57. doi: 10.1038/1711 CrossRefPubMedGoogle Scholar
  25. Parera MC, van Dooren M, van Kempen M, de Krijger R, Grosveld F, Tibboel D, Rottier R (2005) Distal angiogenesis: a new concept for lung vascular morphogenesis. Am J Physiol Lung Cell Mol Physiol 288:L141–L149. doi: 10.1152/ajplung.00148.2004 CrossRefPubMedGoogle Scholar
  26. Park HL et al (2000) Mouse Gli1 mutants are viable but have defects in SHH signaling in combination with a Gli2 mutation. Development 127:1593–1605PubMedGoogle Scholar
  27. Pepicelli CV, Lewis PM, McMahon AP (1998) Sonic hedgehog regulates branching morphogenesis in the mammalian lung. Curr Biol: CB 8:1083–1086CrossRefPubMedGoogle Scholar
  28. Pola R et al (2001) The morphogen Sonic hedgehog is an indirect angiogenic agent upregulating two families of angiogenic growth factors. Nat Med 7:706–711. doi: 10.1038/89083 CrossRefPubMedGoogle Scholar
  29. Ren X et al (2014) FOXF1 transcription factor is required for formation of embryonic vasculature by regulating VEGF signaling in endothelial cells. Circ Res 115:709–720. doi: 10.1161/CIRCRESAHA.115.304382 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Shashikant CS, Bieberich CJ, Belting HG, Wang JC, Borbely MA, Ruddle FH (1995) Regulation of Hoxc-8 during mouse embryonic development: identification and characterization of critical elements involved in early neural tube expression. Development 121:4339-4347PubMedGoogle Scholar
  31. Stankiewicz P et al (2009) Genomic and genic deletions of the FOX gene cluster on 16q24.1 and inactivating mutations of FOXF1 cause alveolar capillary dysplasia and other malformations. Am J Hum Genet 84:780–791. doi: 10.1016/j.ajhg.2009.05.005 CrossRefPubMedPubMedCentralGoogle Scholar
  32. Szafranski P et al (2013) Small noncoding differentially methylated copy-number variants, including lncRNA genes, cause a lethal lung developmental disorder. Genome Res 23:23–33. doi: 10.1101/gr.141887.112 CrossRefPubMedPubMedCentralGoogle Scholar
  33. Taya Y, O'Kane S, Ferguson MW (1999) Pathogenesis of cleft palate in TGF-beta3 knockout mice. Development 126:3869–3879PubMedGoogle Scholar
  34. Tsai MC et al (2010) Long noncoding RNA as modular scaffold of histone modification complexes. Science 329:689–693. doi: 10.1126/science.1192002 CrossRefPubMedPubMedCentralGoogle Scholar
  35. van Tuyl M, Post M (2000) From fruitflies to mammals: mechanisms of signalling via the Sonic hedgehog pathway in lung development. Respir Res 1:30–35. doi: 10.1186/rr9 CrossRefPubMedPubMedCentralGoogle Scholar
  36. van Tuyl M, Liu J, Wang J, Kuliszewski M, Tibboel D, Post M (2005) Role of oxygen and vascular development in epithelial branching morphogenesis of the developing mouse lung. Am J Physiol Lung Cell Mol Physiol 288:L167–L178. doi: 10.1152/ajplung.00185.2004 CrossRefPubMedGoogle Scholar
  37. van Tuyl M, Groenman F, Wang J, Kuliszewski M, Liu J, Tibboel D, Post M (2007) Angiogenic factors stimulate tubular branching morphogenesis of sonic hedgehog-deficient lungs. Dev Biol 303:514–526. doi: 10.1016/j.ydbio.2006.11.029 CrossRefPubMedGoogle Scholar
  38. Wang KC et al (2011) A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression. Nature 472:120–124. doi: 10.1038/nature09819 CrossRefPubMedPubMedCentralGoogle Scholar
  39. Wei G et al (2009) Ets1 and Ets2 are required for endothelial cell survival during embryonic angiogenesis. Blood 114:1123–1130. doi: 10.1182/blood-2009-03-211391 CrossRefPubMedPubMedCentralGoogle Scholar
  40. White AC, Xu J, Yin Y, Smith C, Schmid G, Ornitz DM (2006) FGF9 and SHH signaling coordinate lung growth and development through regulation of distinct mesenchymal domains. Development 133:1507–1517. doi: 10.1242/dev.02313 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Hyejin Seo
    • 1
    • 2
  • Jinsun Kim
    • 1
    • 2
  • Gi-Hee Park
    • 1
  • Yuri Kim
    • 1
    • 2
  • Sung-Won Cho
    • 1
    Email author
  1. 1.Division of Anatomy and Developmental Biology, Department of Oral BiologyYonsei University College of DentistrySeoulKorea
  2. 2.BK21 PLUS ProjectYonsei University College of DentistrySeoulKorea

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