title: Transcriptome analyses based on genetic screens for Pax3 myogenic targets in the mouse embryo.
abstract: BACKGROUND
  Pax3 is a key upstream regulator of the onset of myogenesis, controlling progenitor cell survival and behaviour as well as entry into the myogenic programme. It functions in the dermomyotome of the somite from which skeletal muscle derives and in progenitor cell populations that migrate from the somite such as those of the limbs. Few Pax3 target genes have been identified. Identifying genes that lie genetically downstream of Pax3 is therefore an important endeavour in elucidating the myogenic gene regulatory network.
  
  
  RESULTS
  We have undertaken a screen in the mouse embryo which employs a Pax3GFP allele that permits isolation of Pax3 expressing cells by flow cytometry and a Pax3PAX3-FKHR allele that encodes PAX3-FKHR in which the DNA binding domain of Pax3 is fused to the strong transcriptional activation domain of FKHR. This constitutes a gain of function allele that rescues the Pax3 mutant phenotype. Microarray comparisons were carried out between Pax3GFP/+ and Pax3GFP/PAX3-FKHR preparations from the hypaxial dermomyotome of somites at E9.5 and forelimb buds at E10.5. A further transcriptome comparison between Pax3-GFP positive and negative cells identified sequences specific to myogenic progenitors in the forelimb buds. Potential Pax3 targets, based on changes in transcript levels on the gain of function genetic background, were validated by analysis on loss or partial loss of function Pax3 mutant backgrounds. Sequences that are up- or down-regulated in the presence of PAX3-FKHR are classified as somite only, somite and limb or limb only. The latter should not contain sequences from Pax3 positive neural crest cells which do not invade the limbs. Verification by whole mount in situ hybridisation distinguishes myogenic markers. Presentation of potential Pax3 target genes focuses on signalling pathways and on transcriptional regulation.
  
  
  CONCLUSIONS
  Pax3 orchestrates many of the signalling pathways implicated in the activation or repression of myogenesis by regulating effectors and also, notably, inhibitors of these pathways. Important transcriptional regulators of myogenesis are candidate Pax3 targets. Myogenic determination genes, such as Myf5 are controlled positively, whereas the effect of Pax3 on genes encoding inhibitors of myogenesis provides a potential brake on differentiation. In the progenitor cell population, Pax7 and also Hdac5 which is a potential repressor of Foxc2, are subject to positive control by Pax3.
authors.affiliation: CNRS URA 2578, Département de Biologie du Développement, Institut Pasteur, 25 Rue du Dr Roux, Paris, France.
authors.forename: Mounia
authors.initials: M
authors.lastname: Lagha
authors.forename: Takahiko
authors.initials: T
authors.lastname: Sato
authors.forename: Béatrice
authors.initials: B
authors.lastname: Regnault
authors.forename: Ana
authors.initials: A
authors.lastname: Cumano
authors.forename: Aimée
authors.initials: A
authors.lastname: Zuniga
authors.forename: Jonathan
authors.initials: J
authors.lastname: Licht
authors.forename: Frédéric
authors.initials: F
authors.lastname: Relaix
authors.forename: Margaret
authors.initials: M
authors.lastname: Buckingham
mesh_terms: D000818:Animals; D001483:Base Sequence; D002470:Cell Survival; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism*; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D018507:Gene Expression Regulation, Developmental; D005820:Genetic Testing; D006655:Histone Deacetylases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D024510:Muscle Development / Q000235:genetics*; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D000072041:PAX3 Transcription Factor; D051763:PAX7 Transcription Factor / Q000235:genetics / Q000378:metabolism; D051761:Paired Box Transcription Factors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; C467200:Foxo1 protein, mouse; C491868:Lbx1h protein, mouse; D009124:Muscle Proteins; D000072041:PAX3 Transcription Factor; D051763:PAX7 Transcription Factor; D051761:Paired Box Transcription Factors; C497322:Pax7 protein, mouse; D012333:RNA, Messenger; C068686:Pax3 protein, mouse; C498618:Hdac5 protein, mouse; D006655:Histone Deacetylases