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Combinatorial activity of Six1-2-4 genes in cephalic neural crest cells controls craniofacial and brain development

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Abstract

The combinatorial expression of Hox genes is an evolutionarily ancient program underlying body axis patterning in all Bilateria. In the head, the neural crest (NC)––a vertebrate innovation that contributes to evolutionarily novel skeletal and neural features––develops as a structure free of Hox-gene expression. The activation of Hoxa2 in the Hox-free facial NC (FNC) leads to severe craniofacial and brain defects. Here, we show that this condition unveils the requirement of three Six genes, Six1, Six2, and Six4, for brain development and morphogenesis of the maxillo-mandibular and nasofrontal skeleton. Inactivation of each of these Six genes in FNC generates diverse brain defects, ranging from plexus agenesis to mild or severe holoprosencephaly, and entails facial hypoplasia or truncation of the craniofacial skeleton. The triple silencing of these genes reveals their complementary role in face and brain morphogenesis. Furthermore, we show that the perturbation of the intrinsic genetic FNC program, by either Hoxa2 expression or Six gene inactivation, affects Bmp signaling through the downregulation of Bmp antagonists in the FNC cells. When upregulated in the FNC, Bmp antagonists suppress the adverse skeletal and cerebral effects of Hoxa2 expression. These results demonstrate that the combinatorial expression of Six1, Six2, and Six4 is required for the molecular programs governing craniofacial and cerebral development. These genes are crucial for the signaling system of FNC origin, which regulates normal growth and patterning of the cephalic neuroepithelium. Our results strongly suggest that several congenital craniofacial and cerebral malformations could be attributed to Six genes’ misregulation.

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References

  1. Gans C, Northcutt RG (1983) Neural crest and the origin of vertebrates: a new head. Science 20:268–274. doi:10.1126/science.220.4594.268

    Article  Google Scholar 

  2. Hall BK (1998) Germ layers and the germ-layer theory revisited: primary and secondary germ layers, neural crest as a fourth germ layer, homology, and demise of the germ-layer theory. Evol Biol 30:121–186

    Article  Google Scholar 

  3. Le Douarin NM (1982) The neural crest. Cambridge University Press, Cambridge

    Google Scholar 

  4. Le Douarin NM, Kalcheim C (1999) The neural crest, 2nd edn. Cambridge University Press, Cambridge

    Book  Google Scholar 

  5. Creuzet S, Schuler B, Couly G, Le Douarin NM (2004) Reciprocal relationships between Fgf8 and neural crest cells in development. Proc Natl Acad Sci USA 101:4843–4847. doi:10.1073/pnas.0400869101

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Creuzet SE, Martinez S, Le Douarin NM (2006) The cephalic neural crest exerts a critical effect on forebrain and midbrain development. Proc Natl Acad Sci USA 103:1433–1438. doi:10.1073/pnas.0605899103

    Article  Google Scholar 

  7. Shimamura K, Rubenstein JL (1997) Inductive interactions direct early regionalization of the mouse forebrain. Development 124:2709–2718 ISSN: 0950–1991

    CAS  PubMed  Google Scholar 

  8. Martinez S, Crossley PH, Cobos I, Rubenstein JLR, Martin GR (1999) FGF8 induces formation of an ectopic isthmic organizer and isthmocerebellar development via a repressive effect on Otx2 expression. Development 126:1189–1200 ISSN: 0950–1991

    CAS  PubMed  Google Scholar 

  9. Echevarría D, Vieira C, Gimeno L, Martínez S (2003) Neuroepithelial secondary organizers and cell fate specification in the developing brain. Brain Res Rev 43:179–191. doi:10.1016/j.brainresrev.2003.08.002

    Article  PubMed  Google Scholar 

  10. Creuzet SE (2009) Regulation of pre-otic brain development by the cephalic neural crest. Proc Natl Acad Sci USA 106:15774–15779. doi:10.1073/pnas.0906072106

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Ohkubo Y, Chiang C, Rubenstein JLR (2002) Coordinate regulation and synergistic actions of BMP4, SHH and FGF8 in the rostral prosencephalon regulate morphogenesis of the telencephalic and optic vesicles. Neuroscience 111:1–17. doi:10.1016/S0306-4522(01)00616-9

    Article  CAS  PubMed  Google Scholar 

  12. Creuzet S, Couly G, Bennaceur S, Vincent C, Le Douarin NM (2002) Negative effect of hox gene expression on the development of the neural crest-derived facial skeleton. Development 129:4301–4313 ISSN: 0950–1991

    CAS  PubMed  Google Scholar 

  13. Kutejova E, Engist B, Mallo M, Kanzler B, Bobola N (2005) Hoxa2 downregulates Six2 in the neural crest-derived mesenchyme. Development 132:469–478. doi:10.1242/dev.01536

    Article  CAS  PubMed  Google Scholar 

  14. Le Douarin N, Dieterlen-Lièvre F, Creuzet S, Teillet M-A (2008) Quail––chick transplantations. Methods Cell Biol 87:19–58. doi:10.1016/S0091-679X(08)00202-1

    Article  PubMed  Google Scholar 

  15. Grapin-Botton A, Bonnin M-A, McNaughton LA, Krumlauf R, Le Douarin NM (1995) Plasticity of transposed rhombomeres: Hox gene induction is correlated with phenotypic modifications. Development 121:2707–2721 ISSN: 0950–1991

    CAS  PubMed  Google Scholar 

  16. Pekarik V, Bourikas D, Miglino N, Joset P, Stoekli E (2003) Screening for gene function in chicken embryo using RNAi and electroporation. Nat Biotech 21:93–96. doi:10.1038/nbt770

    Article  CAS  Google Scholar 

  17. Stoeckli ET (2003) RNAi in avian embryos. In RNAi: a guide to gene silencing, 297–312. Cold Spring Harbor Laboratory, New York. ISBN 978-087969704-4

  18. Self M, Lagutin OV, Bowling B, Hendrix J, Cai Y, Dressler GR, Oliver G (2006) Six2 is required for suppression of nephrogenesis and progenitor renewal in the developing kidney. EMBO J 25:5214–5228. doi:10.1038/sj.emboj.7601381

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  19. Petrovová E, Sedmera D, Míšek I, Lešník F, Luptáková L (2009) Bendiocarbamate toxicity in the chick embryo. Folia Biol (Praha) 55:61–65 ISSN: 0015–5500

    Google Scholar 

  20. Henrique D, Adam J, Myat A, Chitnis A, Lewis J, Ish-Horowicz D (1995) Expression of a delta homologue in prospective neurons in the chick. Nature 375:787–790. doi:10.1038/375787a0

    Article  CAS  PubMed  Google Scholar 

  21. Crossley PH, Martinez S, Martin GR (1996) Midbrain development induced by FGF8 in the chick embryo. Nature 380:66–68. doi:10.1038/380066a0

    Article  CAS  PubMed  Google Scholar 

  22. Riddle RD, Johnson RL, Laufer E, Tabin CJ (1993) Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 25:1401–1416. doi:10.1016/0092-8674(93)90626-2

    Article  Google Scholar 

  23. Francis PH, Richardson MK, Brickell PM, Tickle C (1994) Bone morphogenetic proteins and a signaling pathway that controls patterning in the developing chick limb. Development 120:209–218 ISSN: 0950–1991

    CAS  PubMed  Google Scholar 

  24. Connolly DJ, Patel K, Cooke J (1997) Chick noggin is expressed in the organizer and neural plate during axial development, but offers no evidence of involvement in primary axis formation. Int J Dev Biol 41:389–396 ISSN: 0214–6282

    CAS  PubMed  Google Scholar 

  25. Garda AL, Puelles L, Rubenstein JLR (2002) Expression patterns of Wnt8b and Wnt7b in the chicken embryonic brain suggest a correlation with forebrain patterning centers and morphogenesis. Neuroscience 113:689–698. doi:10.1016/S0306-4522(02)00171-9

    Article  CAS  PubMed  Google Scholar 

  26. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucl Acids Res 29:e45. doi:10.1093/nar/29.9.e45

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. Lako M, Lindsay S, Bullen P, Wilson DI, Robson SC, Strachan T (1998) A novel mammalian Wnt gene, WNT8B, shows brain-restricted expression in early development, with sharply delimited expression boundaries in the developing forebrain. Hum Mol Gen 7:813–822. doi:10.1093/hmg/7.5.813

    Article  CAS  PubMed  Google Scholar 

  28. Tzahor E, Kempf H, Mootoosamy RC, Poon AC, Abzhanov A, Tabin CJ, Dietrich S, Lassar AB (2003) Antagonists of Wnt and BMP signaling promote the formation of vertebrate head muscle. Gen Dev 17:3087–3099. doi:10.1093/hmg/7.5.813

    Article  CAS  Google Scholar 

  29. Hsu DR, Economides AN, Wang X, Eimon PM, Harland RM (1998) The Xenopus dorsalizing factor gremlin identifies a novel family of secreted proteins that antagonize BMP activities. Mol Cell 1:673–683. doi:10.1016/S1097-2765(00)80067-2

    Article  CAS  PubMed  Google Scholar 

  30. Eimon PM, Harland RM (2001) Xenopus dan, a member of the dan gene family of BMP antagonists, is expressed in derivatives of the cranial and trunk neural crest. Mech Dev 107:187–189. doi:10.1016/S0925-4773(01)00462-2

    Article  CAS  PubMed  Google Scholar 

  31. Wright E, Hargrave MR, Christiansen J, Cooper L, Kun J, Evans T, Gangadharan U, Greenfield A, Koopman P (1995) The Sry-related gene Sox9 is expressed during chondrogenesis in mouse embryos. Nat Genet 9:15–20. doi:10.1038/ng0195-15

    Article  CAS  PubMed  Google Scholar 

  32. Kutejova E, Engist B, Self M, Oliver G, Kirikenko P, Bobola N (2008) Six2 functions redundantly immediately downstream of Hoxa2. Development 135:1463–1470. doi:10.1242/dev.017624

    Article  CAS  PubMed  Google Scholar 

  33. Fairbridge NA, Nicholas A, Dawe CE, Niri FH, Kooistra MK, King-Jones K, McDermid HE (2010) Cecr2 mutations causing exencephaly trigger misregulation of mesenchymal/ectodermal transcription factors. Birth Def Res A 88:619–625. doi:10.1002/bdra.20695

    Article  CAS  Google Scholar 

  34. Kumar JP (2009) The sine oculis homeobox (SIX) family of transcription factors as regulators of development and disease. Cell Mol Life Sci 66:565–583. doi:10.1007/s00018-008-8335-4

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Christophorou NA, Bailey AP, Hanson S, Streit A (2009) Activation of Six1 target genes is required for sensory placode formation. Dev Biol 336:327–336. doi:10.1016/j.ydbio.2009.09.025

    Article  CAS  PubMed  Google Scholar 

  36. Suzuki Y, Keiko I, Kawakami K (2011) Development of gustatory papilla in the absence of Six1 and Six4. J Anat 219:710–721. doi:10.1111/j.1469-7580.2011.01435.x

    Article  PubMed Central  PubMed  Google Scholar 

  37. Fogelgren B, Kuroyama MC, McBratney-Owen B, Spence AA, Malahn LE, Anawati M, Cabatbat C, Alarcon VB, Marikawa Y, Lozanoff S (2008) Misexpression of Six2 is associated with heritable frontonasal dysplasia and renal hypoplasia in 3H1 Br mice. Dev Dyn 237:1767–1779. doi:10.1002/dvdy.21587

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  38. Gendron-Maguire M, Mallo M, Zhang M, Gridley T (1993) Hoxa2 mutant mice exhibit homeotic transformation of skeletal elements derived from cranial neural crest. Cell 75:1317–1331. doi:10.1016/0092-8674(93)90619-2

    Article  CAS  PubMed  Google Scholar 

  39. Rijli F, Mark M, Lakkaraju S, Dierich A, Dolle P, Chambon P (1993) A homeotic transformation is generated in the rostral branchial region of the head by disruption of Hoxa2, which acts as a selector gene. Cell 75:1333–1349. doi:10.1016/0092-8674(93)90620-6

    Article  CAS  PubMed  Google Scholar 

  40. Kanzler B, Kuschert SJ, Liu YH, Mallo M (1998) Hoxa-2 restricts the chondrogenic domain and inhibits bone formation during development of the branchial area. Development 125:2587–2597 ISSN: 0950–1991

    CAS  PubMed  Google Scholar 

  41. Barrow JR, Capecchi MR (1999) Compensatory defects associated with mutations in Hoxa1 restore normal palatogenesis to Hoxa2 mutants. Development 126:5011–5026 ISSN: 0950–1991

    CAS  PubMed  Google Scholar 

  42. Bobola N, Carapuco M, Ohnemus S, Kanzler B, Leibbrandt A, Neubuser A, Drouin J, Mallo M (2003) Mesenchymal patterning by Hoxa2 requires blocking Fgf-dependent activation of Pitx1. Development 130:3403–3414. doi:10.1242/dev.00554

    Article  CAS  PubMed  Google Scholar 

  43. Trumpp A, Depew MJ, Rubenstein JL, Bishop JM, Martin GR (1999) Cre-mediated gene inactivation demonstrates that FGF8 is required for cell survival and patterning of the first branchial arch. Gen Dev 13:3136–3148. doi:10.1101/gad.13.23.3136

    Article  CAS  Google Scholar 

  44. Abu-Issa R, Smyth G, Smoak I, Yamamura K, Meyers EN (2002) Fgf8 is required for pharyngeal arch and cardiovascular development in the mouse. Development 129:4613–4625 ISSN: 0950–1991

    CAS  PubMed  Google Scholar 

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Acknowledgments

We thank Dr. Françoise Dieterlen for critical reading of the manuscript, Drs. Andréa Trentin and Jörg Kobarg for the technical help with qRT-PCR, Drs. Brickell, Monsoro-Burq, Dressler, Oliver, and Niswander for providing plasmids. This work was supported by the Centre National de la Recherche Scientifique, and grants from the Agence Nationale de la Recherche (ANR-BLAN–0153) and the Ecole des Neurosciences de Paris (Réseau Technologique de Recherche Avancée:-Stem Cells). R.C.G. held fellowships from Fondation des Treilles, Egide, Conselho Nacional de Desenvolvimento Científico e Tecnológico/Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CNPq/CAPES––Brazil).

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  1. Ricardo C. Garcez

    Present address: Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, SC, 88040-900, Brazil

Authors and Affiliations

  1. Institut de Neurobiologie, Laboratoire Neurobiologie et Développement, CNRS-UPR3294, avenue de la Terrasse, 91198, Gif-sur-Yvette, France

    Ricardo C. Garcez & Sophie E. Creuzet

  2. Académie des Sciences, 23, quai Conti, 75006, Paris, France

    Nicole M. Le Douarin

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  1. Ricardo C. Garcez
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  2. Nicole M. Le Douarin
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  3. Sophie E. Creuzet
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Correspondence to Sophie E. Creuzet.

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Garcez, R.C., Le Douarin, N.M. & Creuzet, S.E. Combinatorial activity of Six1-2-4 genes in cephalic neural crest cells controls craniofacial and brain development. Cell. Mol. Life Sci. 71, 2149–2164 (2014). https://doi.org/10.1007/s00018-013-1477-z

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