Skip to main content
SpringerLink
Log in

Transgenic mice expressing PAX3-FKHR have multiple defects in muscle development, including ectopic skeletal myogenesis in the developing neural tube

  • Original paper
  • Published:
Transgenic Research Aims and scope Submit manuscript

Abstract

The t(2;13) chromosomal translocation is found in the majority of human alveolar rhabdomyosarcomas (RMS). The resulting PAX3-FKHR fusion protein contains PAX3 DNA-binding domains fused to the potent transactivation domain of FKHR, suggesting that PAX3-FKHR functions to deregulate PAX3-specific target genes and signaling pathways. We previously developed transgenic mice expressing PAX3-FKHR under the control of mouse Pax3 regulatory sequences to test this hypothesis. We reported that PAX3-FKHR interferes with normal Pax3 developmental functions, with mice exhibiting neural tube and neural crest abnormalities that mimic those found in Pax3-deficient Splotch mice. Here we expanded those studies to show that developmental expression of PAX3-FKHR results in aberrant myogenesis in the developing somites and neural tube, leading to ectopic skeletal muscle formation in the mature spinal cord. Gene expression profiling indicated that PAX3-FKHR expression in the developing neural tube induces a myogenic pattern of gene expression at the expense of the normal neurogenic program. Somite defects in PAX3-FKHR transgenic animals resulted in skeletal malformations that included rib fusions and mis-attachments. As opposed to the neural tube defects, the severity of the rib phenotype was rescued by reducing Pax3 levels through mating with Splotch mice. Embryos from the transgenic line expressing the highest levels of PAX3-FKHR had severe neural tube defects, including exencephaly, and almost half of the embryos died between gestational ages E13.5-E15.5. Nearly all of the embryos that survived to term died after birth due to severe spina bifida, rather than the absence of a muscular diaphragm. These studies reveal a prominent role for PAX3-FKHR in disrupting Pax3 functions and in deregulating skeletal muscle development, suggesting that this fusion protein plays a critical role in the pathogenesis of␣alveolar RMS by influencing the commitment␣and differentiation of the myogenic cell lineage.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Anastasi S, Giordano S, Sthandier O, Gambarotta G, Maione R, Comoglio P, Amati P (1997) A natural hepatocyte growth factor/scatter factor autocrine loop in myoblast cells and the effect of the constitutive Met kinase activation on myogenic differentiation. J Cell Biol 137:1057–1068

    Article  PubMed  CAS  Google Scholar 

  • Anderson MJ, Shelton GD, Cavenee WK, Arden KC (2001) Embryonic expression of the tumor-associated PAX3-FKHR fusion protein interferes with the developmental functions of Pax3. Proc Natl Acad Sci USA 98:1589–1594

    Article  PubMed  CAS  Google Scholar 

  • Anderson MJ, Viars CS, Czekay S, Cavenee WK, Arden KC (1998) Cloning and characterization of three human forkhead genes that comprise an FKHR-like gene subfamily. Genomics 47:187–199

    Article  PubMed  CAS  Google Scholar 

  • Arden KC, Anderson MJ, Finckenstein FG, Czekay S, Cavenee WK (1996) Detection of the t(2;13) chromosomal translocation in alveolar rhabdomyosarcoma using the reverse transcriptase-polymerase chain reaction. Genes Chromosomes Cancer 16:254–260

    Article  PubMed  CAS  Google Scholar 

  • Auerbach R (1954) Analysis of the developmental effects of a lethal mutation in the house mouse. J Exp Zool 127:305–329

    Article  Google Scholar 

  • Bennicelli JL, Fredericks WJ, Wilson RB, Rauscher FJ III, Barr FG (1995) Wild type PAX3 protein and the PAX3-FKHR fusion protein of alveolar rhabdomyosarcoma contain potent, structurally distinct transcriptional activation domains. Oncogene 11:119–130

    PubMed  CAS  Google Scholar 

  • Braun T, Rudnicki MA, Arnold H-H, Jaenisch R (1992) Targeted inactivation of the muscle regulatory gene Myf-5 results in abnormal rib development and perinatal death. Cell 71:369–382

    Article  PubMed  CAS  Google Scholar 

  • Brown CB, Engleka KA, Wenning J, Min Lu M, Epstein JA (2005) Identification of a hypaxial somite enhancer element regulating Pax3 expression in migrating myoblasts and characterization of hypaxial muscle Cre transgenic mice. Genesis 41:202–209

    Article  PubMed  CAS  Google Scholar 

  • Conlon R (1994) In situ hybridization of whole mount embryos with RNA probes. In: Hogan B, Beddington R, Costantini F, Lacy E (eds) Manipulating the mouse embryo: a laboratory manual, 2nd edn. Cold␣Spring Harbor Laboratory Press, New York, pp␣360–362

    Google Scholar 

  • Conway SJ, Henderson DJ, Kirby ML, Anderson RH, Copp AJ (1997) Development of a lethal congenital heart defect in the splotch (Pax3) mutant mouse. Cardiovascular Res 36:163–173

    Article  CAS  Google Scholar 

  • Daston G, Lamar E, Olivier M, Goulding M (1996) Pax-3 is necessary for migration but not differentiation of limb muscle precursors in the mouse. Development 122:1017–1027

    PubMed  CAS  Google Scholar 

  • Davis RJ, D’Cruz CM, Lovell MA, Biegel JA, Barr FG (1994) Fusion of PAX7 to FKHR by the variant t(1;13)(p36;q14) translocation in alveolar rhabdomyosarcoma. Cancer Res 54:2869–2872

    PubMed  CAS  Google Scholar 

  • Delfini M-C, Duprez D (2004) Ectopic Myf5 or MyoD prevents the neuronal differentiation program in addition to inducing skeletal muscle differentiation, in the chick neural tube. Development 131:713–723

    Article  PubMed  CAS  Google Scholar 

  • Epstein JA, Lam P, Jepeal L, Maas RL, Shapiro DN (1995) Pax3 inhibits myogenic differentiation of cultured myoblast cells. J Biol Chem 270:11719–11722

    Article  PubMed  CAS  Google Scholar 

  • Epstein JA, Shapiro DN, Cheng J, Lam PYP, Maas RL (1996) Pax3 modulates expression of the c-Met receptor during limb muscle development. Proc Natl Acad Sci USA 93:4213–4218

    Article  PubMed  CAS  Google Scholar 

  • Ferracini R, Olivero M, Di Renzo MF, Martano M, De Giovanni C, Nanni P, Basso G, Scotlandi K, Lollini P-L, Comoglio PM (1996) Retrogenic expression of the MET proto-oncogene correlates with the invasive phenotype of human rhabdomyosarcomas. Oncogene 12:1697–1705

    PubMed  CAS  Google Scholar 

  • Franz T (1989) Persistent truncus arteriosus in the splotch mutant mouse. Anat Embryol 180:457–464

    Article  PubMed  CAS  Google Scholar 

  • Fredericks WJ, Galili N, Mukhopadhyay S, Rovera G, Bennicelli J, Barr FG, Rauscher FJ III (1995) The PAX3-FKHR fusion protein created by the t(2;13) translocation in alveolar rhabdomyosarcomas is a more potent transcriptional activator than PAX3. Mol Cell Biol 15:1522–1535

    PubMed  CAS  Google Scholar 

  • Galili N, Davis RJ, Fredericks WJ, Mukhopadhyay S, Rauscher FJ III, Emanuel BS, Rovera G, Barr FG (1993) Fusion of a fork head domain gene to PAX3 in the solid tumour alveolar rhabdomyosarcoma. Nat Genet 5:230–235

    Article  PubMed  CAS  Google Scholar 

  • Garrick D, Fiering S, Martin DIK, Whitelaw E (1998) Repeat-induced gene silencing in mammals. Nat Genet 18:56–59

    Article  PubMed  CAS  Google Scholar 

  • Goulding MD, Chalepakis G, Deutsch U, Erselius JR, Gruss P (1991) Pax-3, a novel murine DNA binding protein expressed during early neurogenesis. EMBO J 10:1135–1147

    PubMed  CAS  Google Scholar 

  • Hasty P, Bradley A, Morris JH, Edmondson DG, Venuti JM, Olson EN, Klein WH (1993) Muscle deficiency and neonatal death in mice with a targeted mutation in the myogenin gene. Nature 364:501–506

    Article  PubMed  CAS  Google Scholar 

  • Hosack DA, Dennis G Jr, Sherman BT, Lane HC, Lempicki RA (2003) Identifying biological themes within lists of genes with EASE. Genome Biol 4: R70

  • James AC, Veitch JG, Zareh AR, Triche T (2004) Sensitivity and specificity of five abundance estimators for high-density oligonucleotide microarrays. Bioinformatics 20:1060–1065

    Article  PubMed  CAS  Google Scholar 

  • Kablar B, Rudnicki MA (1999) Development in the absence of skeletal muscle results in the sequential ablation of motor neurons from the spinal cord to the brain. Dev Biol 208:93–109

    Article  PubMed  CAS  Google Scholar 

  • Keller C, Arenkiel BR, Coffin CM, El-Bardeesy N, DePinho RA, Capecchi MR (2004a) Alveolar rhabdomyosarcomas in conditional Pax3:Fkhr mice: cooperativity of Ink4a/ARF and Trp53 loss of function. Genes Dev 18:2614–2626

    Article  CAS  Google Scholar 

  • Keller C, Hansen MS, Coffin CM, Capecchi MR (2004b) Pax3:Fkhr interferes with embryonic Pax3 and Pax7 function: implications for alveolar rhabdomyosarcoma cell of origin. Genes Dev 18:2608–2613

    Article  CAS  Google Scholar 

  • Khan J, Bittner ML, Saal LH, Teichmann U, Azorsa DO, Gooden GC, Pavan WJ, Trent JM, Meltzer PS (1999) cDNA microarrays detect activation of a myogenic transcription program by the PAX3-FKHR fusion oncogene. Proc Natl Acad Sci USA 96:13264–13269

    Article  PubMed  CAS  Google Scholar 

  • Lagutina I, Conway SJ, Sublett J, Grosveld GC (2002) Pax3-FKHR knock-in mice show developmental aberrations but do not develop tumors. Mol Cell Biol 22:7204–7216

    Article  PubMed  CAS  Google Scholar 

  • Lam PYP, Sublett JE, Hollenbach AD, Roussel MF (1999) The oncogenic potential of the Pax3-FKHR fusion protein requires the Pax3 homeodomain recognition helix but not the Pax3 paired-box DNA binding domain. Mol Cell Biol 19:594–601

    PubMed  CAS  Google Scholar 

  • Liu LN, Dias P, Houghton PJ (1998) Mutation of Thr115 in MyoD positively regulates function in murine fibroblasts and human rhabdomyosarcoma cells. Cell Growth Diff 9:699–711

    PubMed  CAS  Google Scholar 

  • Milewski RC, Chi NC, Li J, Brown C, Lu MM, Epstein JA (2004) Identification of minimal enhancer elements sufficient for Pax3 expression in neural crest and implication of Tead2 as a regulator of Pax3. Development 131:829–837

    Article  PubMed  CAS  Google Scholar 

  • Natoli TA, Ellsworth MK, Wu C, Gross KW, Pruitt SC (1997) Positive and negative DNA sequence elements are required to establish the pattern of Pax3 expression. Development 124:617–626

    PubMed  CAS  Google Scholar 

  • Peter M, Gilbert E, Delattre O (2001) A multiplex real-time PCR assay for the detection of gene fusions observed in solid tumors. Lab Invest 81:905–912

    PubMed  CAS  Google Scholar 

  • Relaix F, Polimeni M, Rocancourt D, Ponzetto C, Schafer BW, Buckingham M (2003) The transcriptional activator PAX3-FKHR rescues the defects of Pax3 mutant mice but induces a myogenic gain-of-function phenotype with ligand-independent activation of Met signaling in vivo. Genes Dev 17:2950–2965

    Article  PubMed  CAS  Google Scholar 

  • Sah VP, Attardi LD, Mulligan GJ, Williams BO, Bronson RT, Jacks T (1995) A subset of p53-deficient embryos exhibit exencephaly. Nat Genet 10:175–180

    Article  PubMed  CAS  Google Scholar 

  • Sassoon DA, Lyons G, Wright WE, Lin V, Lassar A, Weintraub H, Buckingham M (1989) Expression of two myogenic regulatory factors myogenin and MyoD1 during mouse embryogenesis. Nature 341:303–307

    Article  PubMed  CAS  Google Scholar 

  • Sorensen PHB, Lynch JC, Qualman SJ, Tirabosco R, Lim JF, Maurer HM, Bridge JA, Crist WM, Triche TJ, Barr FG (2002) PAX3-FKHR and PAX7-FKHR gene fusions are prognostic indicators in alveolar rhabdomyosarcoma: a report from the Children’s Oncololgy Group. J Clin Oncol 20:2672–2679

    Article  PubMed  CAS  Google Scholar 

  • Sublett JE, Jeon I-S, Shapiro DN (1995) The alveolar rhabdomyosarcoma PAX3/FKHR fusion protein is a transcriptional activator. Oncogene 11:545–552

    PubMed  CAS  Google Scholar 

  • Takayama H, LaRochelle WJ, Anver M, Bockman DE, Merlino G (1996) Scatter factor/hepatocyte growth factor as a regulator of skeletal muscle and neural crest development. Proc Natl Acad Sci USA 93:5866–5871

    Article  PubMed  CAS  Google Scholar 

  • Tapscott SJ, Thayer MJ, Weintraub H (1993) Deficiency in rhabdomyosarcomas of a factor required for MyoD activity and myogenesis. Science 259:1450–1453

    Article  PubMed  CAS  Google Scholar 

  • Tremblay P, Dietrich S, Mericskay M, Schubert FR, Li Z, Paulin D (1998) A crucial role for Pax3 in the development of the hypaxial musculature and the long-range migration of muscle precursors. Dev Biol 203:49–61

    Article  PubMed  CAS  Google Scholar 

  • Triche TJ, Sorensen PHB (2002) Molecular pathology of pediatric malignancies. In: Pizzo PA, Poplack DG (eds) Principles and practice of pediatric oncology, 4th edn. Lippincott Williams & Wilkins, Philadelphia, pp 161–204

    Google Scholar 

  • Wilkinson DG (1992) Whole mount in situ hybridization of vertebrate embryos. In: Wilkinson DG (eds) In situ hybridization: a practical approach. Oxford University Press, New York, pp 75–83

    Google Scholar 

  • Yamamoto Y, Livet J, Pollock RA, Garces A, Arce V, deLapeyriere O, Henderson CE (1997) Hepatocyte growth factor (HGF/SF) is a muscle-derived survival factor for a subpopulation of embryonic motoneurons. Development 124:2903–2913

    PubMed  CAS  Google Scholar 

  • Yang X-M, Vogan K, Gros P, Park M (1996) Expression of the met receptor tyrosine kinase in muscle progenitor cells in somites and limbs is absent in Splotch mice. Development 122:2163–2171

    PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank Hiroyuki Shimada and Sue Ann Phung for tissue sectioning; Patricia Reid for electron microscopy; Betty Schaub and Sitara Waidyaratne for microarray processing; Andrew Lassar, Morag Park and Peter Gruss for the mouse MyoD, c-met and Pax3 cDNA constructs, respectively; Violette Shahbazian and Antonia Boyer for excellent technical assistance; Diane Shelton, George McNamara and Nicole Tedeschi for helpful discussions. This work was supported by a Faculty Research Career Development Award (MJA) from the Saban Research Institute at Childrens Hospital Los Angeles. WKC is a Fellow of the National Foundation for Cancer Research.

Author information

Authors and Affiliations

  1. Childrens Hospital Los Angeles Research Institute, Los Angeles, CA , 90027, USA

    Friedrich Graf Finckenstein & Michael J. Anderson

  2. Department of Pathology & Laboratory Medicine, University of Southern California Keck School of Medicine, Los Angeles, CA, 90027, USA

    Elai Davicioni & Michael J. Anderson

  3. The Scripps Research Institute, La Jolla, CA, USA

    Kent G. Osborn

  4. Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, CA , 92093-0660, USA

    Webster K. Cavenee & Karen C. Arden

  5. Department of Medicine, University of California at San Diego, La Jolla, CA , 92093-0660, USA

    Webster K. Cavenee & Karen C. Arden

  6. Center for Molecular Genetics, University of California at San Diego, La Jolla, CA , 92093-0660, USA

    Webster K. Cavenee

  7. Cancer Center, University of California at San Diego, La Jolla, CA , 92093-0660, USA

    Webster K. Cavenee & Karen C. Arden

Authors
  1. Friedrich Graf Finckenstein
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Elai Davicioni
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kent G. Osborn
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Webster K. Cavenee
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Karen C. Arden
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Michael J. Anderson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michael J. Anderson.

Electronic Supplementary Material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Finckenstein, F.G., Davicioni, E., Osborn, K.G. et al. Transgenic mice expressing PAX3-FKHR have multiple defects in muscle development, including ectopic skeletal myogenesis in the developing neural tube. Transgenic Res 15, 595–614 (2006). https://doi.org/10.1007/s11248-006-9011-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11248-006-9011-9

Keywords

Search

Navigation