Manipulating the Fragile X Mental Retardation Proteins in the Frog

  • Marc-Etienne Huot
  • Nicolas Bisson
  • Thomas Moss
  • Edouard W. KhandjianEmail author
Part of the Results and Problems in Cell Differentiation book series (RESULTS, volume 54)


The frog is a model of choice to study gene function during early development, since a large number of eggs are easily obtained and rapidly develop external to the mother. This makes it a highly flexible model system in which direct tests of gene function can be investigated by microinjecting RNA antisense reagents. Two members of the Fragile X Related (FXR) gene family, namely xFmr1 and xFxr1 have been identified in Xenopus. While the tissue distribution of their products was found to be identical to that in mammals, the pattern of isoform expression is less complex. Translational silencing of the xFmr1 and xFxr1 mRNAs by microinjection of antisense morpholino oligonucleotides (MO) induced dramatic morphological alterations, revealing tissue-specific requirements for each protein during development and in maintaining the steady state levels of a range of transcripts in these tissues. The power and versatility of the frog model is that the MO-induced phenotypes can be rescued by microinjection of the corresponding MO-insensitive mRNAs. Most importantly, this animal model allows one rapidly to determine whether any member of the FXR family can compensate for the absence of another, an approach that cannot be performed in other animal models.


FMR1 Gene Morpholino Oligonucleotide Muscle Formation Antisense Morpholino Oligonucleotide Cranial Neural Crest Cell 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



Research on the frog model has been supported by grants from the Natural Sciences and Engineering Research Council of Canada, the Canadian Institutes of Health Research, the FRAXA Research Foundation (USA), and the Fragile X Research Foundation of Canada (to E.W.K.) and by a grant from the National Cancer Institute of Canada and with funds from the Canadian Cancer Society (to T.M.).


  1. Antar LN, Afroz R, Dictenberg JB, Carroll RC, Bassell GJ (2004) Metabotropic glutamate receptor activation regulates fragile x mental retardation protein and FMR1 mRNA localization differentially in dendrites and at synapses. J Neurosci 24:2648–2655PubMedCrossRefGoogle Scholar
  2. Bakker CE, Verheij C, Willemsen R, Van der Helm R, Oerlemans F, Vermey M, Bygrave A, Hoogeveen AT, Oostra BA (1994) Fmr1 knockout mice: a model to study fragile X mental retardation. Cell 78:23–33Google Scholar
  3. Bakker CE, de Diego OY, Bontekoe C, Raghoe P, Luteijn T, Hoogeveen AT, Oostra BA, Willemsen R (2000) Immunocytochemical and biochemical characterization of FMRP, FXR1P, and FXR2P in the mouse. Exp Cell Res 258:162–170PubMedCrossRefGoogle Scholar
  4. Bardoni B, Davidovic L, Bensaid M, Khandjian EW (2006) The fragile X syndrome: exploring its molecular basis and seeking a treatment. Expert Rev Mol Med 8:1–16PubMedCrossRefGoogle Scholar
  5. Bechara E, Davidovic L, Melko M, Bensaid M, Tremblay S, Grosgeorge J, Khandjian EW, Lalli E, Bardoni B (2007) Fragile X related protein 1 isoforms differentially modulate the affinity of fragile X mental retardation protein for G-quartet RNA structure. Nucleic Acids Res 35:299–306PubMedCrossRefGoogle Scholar
  6. Bill BR, Petzold AM, Clark KJ, Schimmenti LA, Ekker SC (2009) A primer for morpholino use in zebrafish. Zebrafish 6:69–77PubMedCrossRefGoogle Scholar
  7. Blonden L, van’t Padje S, Severijnen LA, Destree O, Oostra BA, Willemsen R (2005) Two members of the Fxr gene family, Fmr1 and Fxr1, are differentially expressed in Xenopus tropicalis. Int J Dev Biol 49:437–441PubMedCrossRefGoogle Scholar
  8. Bontekoe CJ, McIlwain KL, Nieuwenhuizen IM, Yuva-Paylor LA, Nellis A, Willemsen R, Fang Z, Kirkpatrick L, Bakker CE, McAninch R et al (2002) Knockout mouse model for Fxr2: a model for mental retardation. Hum Mol Genet 11:487–498PubMedCrossRefGoogle Scholar
  9. Coffee RL Jr, Tessier CR, Woodruff EA 3rd, Broadie K (2010) Fragile X mental retardation protein has a unique, evolutionarily conserved neuronal function not shared with FXR1P or FXR2P. Dis Model Mech 3:471–485PubMedCrossRefGoogle Scholar
  10. Cripe L, Morris E, Fulton AB (1993) Vimentin mRNA location changes during muscle development. Proc Natl Acad Sci USA 90:2724–2728PubMedCrossRefGoogle Scholar
  11. Cunningham CL, Martinez Cerdeno V, Navarro E, Prakash A, Angelastro JM, Willemsen R, Hagerman PJ, Pessah IN, Berman RF, Noctor SC (2011) Premutation CGG-repeat expansion of the Fmr1 gene impairs mouse neocortical development. Hum Mol Genet 20:64–79PubMedCrossRefGoogle Scholar
  12. Darnell JC, Jensen KB, Jin P, Brown V, Warren ST, Darnell RB (2001) Fragile X mental retardation protein targets G quartet mRNAs important for neuronal function. Cell 107:489–499PubMedCrossRefGoogle Scholar
  13. Darnell JC, Fraser CE, Mostovetsky O, Darnell RB (2009) Discrimination of common and unique RNA-binding activities among Fragile X mental retardation protein paralogs. Hum Mol Genet 18:3164–3177PubMedCrossRefGoogle Scholar
  14. Davidovic L, Tremblay S, De Koninck P, Khandjian EW (2005) Fragile X mental retardation protein: to be or not to be a translational repressor. In: Sung Y-J, Denman RB (eds) The molecular basis of fragile X syndrome. Research Signpost, Trivandrum, pp 129–143Google Scholar
  15. De Diego OY, Severijnen LA, van Cappellen G, Schrier M, Oostra B, Willemsen R (2002) Transport of fragile X mental retardation protein via granules in neurites of PC12 cells. Mol Cell Biol 22:8332–8341CrossRefGoogle Scholar
  16. Dobkin C, Rabe A, Dumas R, El Idrissi A, Haubenstock H, Brown WT (2000) Fmr1 knockout mouse has a distinctive strain-specific learning impairment. Neuroscience 100:423–429PubMedCrossRefGoogle Scholar
  17. Dube M, Huot ME, Khandjian EW (2000) Muscle specific fragile X related protein 1 isoforms are sequestered in the nucleus of undifferentiated myoblast. BMC Genet 1:4PubMedCrossRefGoogle Scholar
  18. Eisen JS, Smith JC (2008) Controlling morpholino experiments: don’t stop making antisense. Development 135:1735–1743PubMedCrossRefGoogle Scholar
  19. Engels B, van’t Padje S, Blonden L, Severijnen LA, Oostra BA, Willemsen R (2004) Characterization of Fxr1 in Danio rerio: a simple vertebrate model to study costamere development. J Exp Biol 207:3329–3338PubMedCrossRefGoogle Scholar
  20. Fulton AB, Alftine C (1997) Organization of protein and mRNA for titin and other myofibril components during myofibrillogenesis in cultured chicken skeletal muscle. Cell Struct Funct 22:51–58PubMedCrossRefGoogle Scholar
  21. Gessert S, Bugner V, Tecza A, Pinker M, Kuhl M (2010) FMR1/FXR1 and the miRNA pathway are required for eye and neural crest development. Dev Biol 341:222–235PubMedCrossRefGoogle Scholar
  22. Greenspan RJ (2007) An introduction to nervous systems. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  23. Guduric-Fuchs J, Mohrlen F, Frohme M, Frank U (2004) A fragile X mental retardation-like gene in a cnidarian. Gene 343:231–238PubMedCrossRefGoogle Scholar
  24. Huot ME, Bisson N, Davidovic L, Mazroui R, Labelle Y, Moss T, Khandjian EW (2005) The RNA-binding protein fragile X-related 1 regulates somite formation in Xenopus laevis. Mol Biol Cell 16:4350–4361PubMedCrossRefGoogle Scholar
  25. Irwin SA, Galvez R, Greenough WT (2000) Dendritic spine structural anomalies in fragile-X mental retardation syndrome. Cereb Cortex 10:1038–1044PubMedCrossRefGoogle Scholar
  26. Khandjian EW, Corbin F, Woerly S, Rousseau F (1996) The fragile X mental retardation protein is associated with ribosomes. Nat Genet 12:91–93PubMedCrossRefGoogle Scholar
  27. Khandjian EW, Bardoni B, Corbin F, Sittler A, Giroux S, Heitz D, Tremblay S, Pinset C, Montarras D, Rousseau F et al (1998) Novel isoforms of the fragile X related protein FXR1P are expressed during myogenesis. Hum Mol Genet 7:2121–2128PubMedCrossRefGoogle Scholar
  28. Khandjian EW, Huot ME, Tremblay S, Davidovic L, Mazroui R, Bardoni B (2004) Biochemical evidence for the association of fragile X mental retardation protein with brain polyribosomal ribonucleoparticles. Proc Natl Acad Sci USA 101:13357–13362PubMedCrossRefGoogle Scholar
  29. Khandjian EW, Tournier B, Séguin S, Tremblay S, De Koninck P, Davidovic L (2009) RNA granules: functions within presynaptic terminals and postsynaptic spines. In: Squire L (ed) Encyclopedia of neuroscience, vol 8. Elsevier, London, UK, pp 389–395CrossRefGoogle Scholar
  30. Kirkpatrick LL, McIlwain KA, Nelson DL (1999) Alternative splicing in the murine and human FXR1 genes. Genomics 59:193–202PubMedCrossRefGoogle Scholar
  31. Klein SL (1987) The first cleavage furrow demarcates the dorsal-ventral axis in Xenopus embryos. Dev Biol 120:299–304PubMedCrossRefGoogle Scholar
  32. Kooy RF (2003) Of mice and the fragile X syndrome. Trends Genet 19:148–154PubMedCrossRefGoogle Scholar
  33. Lim JH, Luo T, Sargent TD, Fallon JR (2005) Developmental expression of Xenopus fragile X mental retardation-1 gene. Int J Dev Biol 49:981–984PubMedCrossRefGoogle Scholar
  34. Mazroui R, Huot ME, Tremblay S, Filion C, Labelle Y, Khandjian EW (2002) Trapping of messenger RNA by fragile X mental retardation protein into cytoplasmic granules induces translation repression. Hum Mol Genet 11:3007–3017PubMedCrossRefGoogle Scholar
  35. Mientjes EJ, Willemsen R, Kirkpatrick LL, Nieuwenhuizen IM, Hoogeveen-Westerveld M, Verweij M, Reis S, Bardoni B, Hoogeveen AT, Oostra BA et al (2004) Fxr1 knockout mice show a striated muscle phenotype: implications for Fxr1p function in vivo. Hum Mol Genet 13:1291–1302PubMedCrossRefGoogle Scholar
  36. Miyashiro KY, Beckel-Mitchener A, Purk TP, Becker KG, Barret T, Liu L, Carbonetto S, Weiler IJ, Greenough WT, Eberwine J (2003) RNA cargoes associating with FMRP reveal deficits in cellular functioning in Fmr1 null mice. Neuron 37:417–431PubMedCrossRefGoogle Scholar
  37. Morris EJ, Fulton AB (1994) Rearrangement of mRNAs for costamere proteins during costamere development in cultured skeletal muscle from chicken. J Cell Sci 107(Pt 3):377–386PubMedGoogle Scholar
  38. Nieuwkoop PD, Faber J (1956) Normal table of Xenopus Laevis (Daudin): a systematical & chronological survey of the development from the fertilized egg till the end of metamorphosis. North-Holland Publishing Company, AmsterdamGoogle Scholar
  39. O’Donnell WT, Warren ST (2002) A decade of molecular studies of fragile X syndrome. Annu Rev Neurosci 25:315–338PubMedCrossRefGoogle Scholar
  40. Papoulas O, Monzo KF, Cantin GT, Ruse C, Yates JR 3rd, Ryu YH, Sisson JC (2010) dFMRP and Caprin, translational regulators of synaptic plasticity, control the cell cycle at the Drosophila mid-blastula transition. Development 137:4201–4209PubMedCrossRefGoogle Scholar
  41. Schaeffer C, Bardoni B, Mandel JL, Ehresmann B, Ehresmann C, Moine H (2001) The fragile X mental retardation protein binds specifically to its mRNA via a purine quartet motif. EMBO J 20:4803–4813PubMedCrossRefGoogle Scholar
  42. Stefani G, Fraser CE, Darnell JC, Darnell RB (2004) Fragile X mental retardation protein is associated with translating polyribosomes in neuronal cells. J Neurosci 24:7272–7276PubMedCrossRefGoogle Scholar
  43. Summerton J, Weller D (1997) Morpholino antisense oligomers: design, preparation, and properties. Antisense Nucleic Acid Drug Dev 7:187–195PubMedCrossRefGoogle Scholar
  44. Tucker B, Richards R, Lardelli M (2004) Expression of three zebrafish orthologs of human FMR1-related genes and their phylogenetic relationships. Dev Genes Evol 214:567–574PubMedCrossRefGoogle Scholar
  45. Wan L, Dockendorff TC, Jongens TA, Dreyfuss G (2000) Characterization of dFMR1, a Drosophila melanogaster homolog of the fragile X mental retardation protein. Mol Cell Biol 20:8536–8547PubMedCrossRefGoogle Scholar
  46. Weiler IJ, Spangler CC, Klintsova AY, Grossman AW, Kim SH, Bertaina-Anglade V, Khaliq H, de Vries FE, Lambers FA, Hatia F et al (2004) Fragile X mental retardation protein is necessary for neurotransmitter-activated protein translation at synapses. Proc Natl Acad Sci USA 101:17504–17509PubMedCrossRefGoogle Scholar
  47. Zhang Y, O'Connor JP, Siomi MC, Srinivasan S, Dutra A, Nussbaum RL, Dreyfuss G (1995) The fragile X mental retardation syndrome protein interacts with novel homologs FXR1 and FXR2. EMBO J 14:5358–5366PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Marc-Etienne Huot
    • 1
  • Nicolas Bisson
    • 2
  • Thomas Moss
    • 1
  • Edouard W. Khandjian
    • 3
    Email author
  1. 1.Centre de recherche en Cancérologie de l’Université LavalCHUQ-Hôtel-Dieu de QuébecQuébecCanada
  2. 2.Samuel Lunenfeld Research InstituteMount Sinai HospitalTorontoCanada
  3. 3.Centre de recherche Université Laval Robert GiffardUniversité LavalQuébecCanada

Personalised recommendations