Transcriptome analyses based on genetic screens for Pax3 myogenic targets in the mouse embryo

CNRS URA 2578, Département de Biologie du Développement, Institut Pasteur, 25 Rue du Dr Roux, Paris, France.
BMC Genomics (Impact Factor: 3.99). 12/2010; 11(1):696. DOI: 10.1186/1471-2164-11-696
Source: PubMed


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.
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.
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.

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    • "Most information was provided by analyses of cell lines derived from alveolar rhabdomyosarcomas, muscle tumors that are caused by a chromosomal translocation that results in a PAX3-FKHR(FOXO1A) or PAX7-FKHR fusion protein, acting as a strong transcriptional activator (see Robson et al., 2006). Cell death complicates loss-of-function screens in the mouse embryo, but a gain-of-function screen of Pax3-expressing cells (Lagha et al., 2010) revealed genes that are up-or downregulated in somites and forelimbs in the presence of an allele of Pax3 encoding a PAX3-FKHR fusion protein. These include transcription factors and components of signaling pathways known to affect myogenesis, including genes that are involved at different stages in the myogenic progression of a somitic cell (Figure 2B). "
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    ABSTRACT: We discuss the upstream regulators of myogenesis that lead to the activation of myogenic determination genes and subsequent differentiation, focusing on the mouse model. Key upstream genes, such as Pax3 and Pax7, Six1 and Six4, or Pitx2, participate in gene regulatory networks at different sites of skeletal muscle formation. MicroRNAs also intervene, with emerging evidence for the role of other noncoding RNAs. Myogenic determination and subsequent differentiation depend on members of the MyoD family. We discuss new insights into mechanisms underlying the transcriptional activity of these factors.
    Preview · Article · Feb 2014 · Developmental Cell
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    • "This involves coordinated transcriptional and posttranscriptional regulations. Despite advances in the inference of complex transcriptional gene regulatory networks in invertebrate embryos (Busser et al., 2012; Gohlke et al., 2008; Hertzano et al., 2011; Isern et al., 2011; Lagha et al., 2010; Taher et al., 2011), this task remains challenging for early vertebrate embryogenesis. We focus on vertebrate neural crest induction, in which early transcriptional regulators activate a complex developmental network, and in which transcriptome analysis can be combined with in vivo experimental validation. "
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    ABSTRACT: Neural crest development is orchestrated by a complex and still poorly understood gene regulatory network. Premigratory neural crest is induced at the lateral border of the neural plate by the combined action of signaling molecules and transcription factors such as AP2, Gbx2, Pax3 and Zic1. Among them, Pax3 and Zic1 are both necessary and sufficient to trigger a complete neural crest developmental program. However, their gene targets in the neural crest regulatory network remain unknown. Here, through a transcriptome analysis of frog microdissected neural border, we identified an extended gene signature for the premigratory neural crest, and we defined novel potential members of the regulatory network. This signature includes 34 novel genes, as well as 44 known genes expressed at the neural border. Using another microarray analysis which combined Pax3 and Zic1 gain-of-function and protein translation blockade, we uncovered 25 Pax3 and Zic1 direct targets within this signature. We demonstrated that the neural border specifiers Pax3 and Zic1 are direct upstream regulators of neural crest specifiers Snail1/2, Foxd3, Twist1, and Tfap2b. In addition, they may modulate the transcriptional output of multiple signaling pathways involved in neural crest development (Wnt, Retinoic Acid) through the induction of key pathway regulators (Axin2 and Cyp26c1). We also found that Pax3 could maintain its own expression through a positive autoregulatory feedback loop. These hierarchical inductions, feedback loops, and pathway modulations provide novel tools to understand the neural crest induction network.
    Full-text · Article · Dec 2013 · Developmental Biology
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    • "This allele rescues the Pax3 mutant phenotype and we therefore devised a screen incorporating a Pax3GFP allele [21] which permits us to purify the Pax3-positive population by flow cytometry. Comparison of the transcriptome of somites at E9.5 and forelimb buds at E10.5 of Pax3GFP/+ and Pax3GFP/PAX3-FKHR embryos led to the identification of genes that are up- or down-regulated in the presence of FKHR [22]. These included known Pax3 targets and genes such as Foxc2, implicated in cell fate decisions in the dermomyotome [23], which is negatively regulated by Pax3. "
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    ABSTRACT: The paired-box homeodomain transcription factor Pax3 is a key regulator of the nervous system, neural crest and skeletal muscle development. Despite the important role of this transcription factor, very few direct target genes have been characterized. We show that Itm2a, which encodes a type 2 transmembrane protein, is a direct Pax3 target in vivo, by combining genetic approaches and in vivo chromatin immunoprecipitation assays. We have generated a conditional mutant allele for Itm2a, which is an imprinted gene, by flanking exons 2-4 with loxP sites and inserting an IRESnLacZ reporter in the 3' UTR of the gene. The LacZ reporter reproduces the expression profile of Itm2a, and allowed us to further characterize its expression at sites of myogenesis, in the dermomyotome and myotome of somites, and in limb buds, in the mouse embryo. We further show that Itm2a is not only expressed in adult muscle fibres but also in the satellite cells responsible for regeneration. Itm2a mutant mice are viable and fertile with no overt phenotype during skeletal muscle formation or regeneration. Potential compensatory mechanisms are discussed.
    Full-text · Article · May 2013 · PLoS ONE
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