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

<p>Abstract</p> <p>Background</p> <p>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 <it>Pax3 </it>is therefore an important endeavour in elucidating the myogenic gene regulatory network.</p> <p>Results</p> <p>We have undertaken a screen in the mouse embryo which employs a <it>Pax3^GFP</it>allele that permits isolation of Pax3 expressing cells by flow cytometry and a <it>Pax3^PAX3-FKHR</it>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 <it>Pax3 </it>mutant phenotype. Microarray comparisons were carried out between <it>Pax3^GFP/+</it>and <it>Pax3^{GFP/PAX3-FKHR}</it>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 <it>Pax3 </it>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 <it>in situ </it>hybridisation distinguishes myogenic markers. Presentation of potential Pax3 target genes focuses on signalling pathways and on transcriptional regulation.</p> <p>Conclusions</p> <p>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 <it>Myf5 </it>are controlled positively, whereas the effect of <it>Pax3 </it>on genes encoding inhibitors of myogenesis provides a potential brake on differentiation. In the progenitor cell population, <it>Pax7 </it>and also <it>Hdac5 </it>which is a potential repressor of <it>Foxc2</it>, are subject to positive control by <it>Pax3</it>.</p

Duplication and Diversification of the Hypoxia-Inducible IGFBP-1 Gene in Zebrafish

Gene duplication is the primary force of new gene evolution. Deciphering whether a pair of duplicated genes has evolved divergent functions is often challenging. The zebrafish is uniquely positioned to provide insight into the process of functional gene evolution due to its amenability to genetic and experimental manipulation and because it possess a large number of duplicated genes.We report the identification and characterization of two hypoxia-inducible genes in zebrafish that are co-ortholgs of human IGF binding protein-1 (IGFBP-1). IGFBP-1 is a secreted protein that binds to IGF and modulates IGF actions in somatic growth, development, and aging. Like their human and mouse counterparts, in adult zebrafish igfbp-1a and igfbp-1b are exclusively expressed in the liver. During embryogenesis, the two genes are expressed in overlapping spatial domains but with distinct temporal patterns. While zebrafish IGFBP-1a mRNA was easily detected throughout embryogenesis, IGFBP-1b mRNA was detectable only in advanced stages. Hypoxia induces igfbp-1a expression in early embryogenesis, but induces the igfbp-1b expression later in embryogenesis. Both IGFBP-1a and -b are capable of IGF binding, but IGFBP-1b has much lower affinities for IGF-I and -II because of greater dissociation rates. Overexpression of IGFBP-1a and -1b in zebrafish embryos caused significant decreases in growth and developmental rates. When tested in cultured zebrafish embryonic cells, IGFBP-1a and -1b both inhibited IGF-1-induced cell proliferation but the activity of IGFBP-1b was significantly weaker.These results indicate subfunction partitioning of the duplicated IGFBP-1 genes at the levels of gene expression, physiological regulation, protein structure, and biological actions. The duplicated IGFBP-1 may provide additional flexibility in fine-tuning IGF signaling activities under hypoxia and other catabolic conditions

Biodiversity and Phylogenetic Relationships of Novel Bacteriocinogenic Strains Isolated from Animal’s Droppings at the Zoological Garden of Lille, France

International audienceThis study aimed at exploring droppings of animals living in captivity in the zoological garden (Zoo) of Lille (France), as novel sources of bacteriocinogenic strains. A collection of 295 bacterial isolates was constituted from droppings of capybara, alpaca, muntjac, zebra, tapir, rhinoceros, binturong, armadillo, saki monkey and cockatoo. Of 295 isolates, 51 exhibited antagonism against a panel of pathogenic target bacteria like Escherichia coli MC4100, Clostridium perfringens DSM 756 and Salmonella enterica subsp. enterica Newport ATCC6962. Remarkably, within this collection, only 2 Gram-negative bacilli exhibited activity against E. coli MC4100 strain used as target organism. Then, the 16S rDNA sequencing revealed these thereafter cited species, Pediococcus pentosaceus, Weissella cibaria, E. coli, Lactobacillus reuteri, Enterococcus hirae and Enterococcus faecalis. Characterization of this antagonism has revealed 11 strains able producing extracellular protease-sensitive inhibitory compounds. These strains included E. coli ICVB442 and ICVB443, Ent. faecalis ICVB472, ICVB474, ICVB477 ICVB479, ICVB481, ICVB497 and ICVB501 and Ped. pentosaceus ICVB491 and ICVB492. The genomes of the 5 most promising bacteriocinogenic strains were sequenced and analysed with Bagel4 software. Afterwards, this bioinformatics analysis permitted to locate genes encoding bacteriocins like colicin Y (E. coli), enterocin 1071A, enterocin 107 B (Ent. faecalis) and penocin A (Ped. pentosaceus), associating the above-mentioned antibacterial activity of proteinaceous nature to possible production of bacteriocins. All these results enabled us to select different bacteriocinogenic strains for a further characterization in terms of beneficial traits