Wnt/Lef1 signaling acts via Pitx2 to regulate somite myogenesis

AbstractWnt signaling has been implicated in somite, limb, and branchial arch myogenesis but the mechanisms and roles are not clear. We now show that Wnt signaling via Lef1 acts to regulate the number of premyogenic cells in somites but does not regulate myogenic initiation in the limb bud or maintenance in the first or second branchial arch. We have also analysed the function and regulation of a putative downstream transcriptional target of canonical Wnt signaling, Pitx2. We show that loss-of-function of Pitx2 decreases the number of myogenic cells in the somite, whereas overexpression increases myocyte number particularly in the epaxial region of the myotome. Increased numbers of mitotic cells were observed following overexpression of Pitx2 or an activated form of Lef1, suggesting an effect on cell proliferation. In addition, we show that Pitx2 expression is regulated by canonical Wnt signaling in the epaxial somite and second branchial arch, but not in the limb or the first branchial arch. These results suggest that Wnt/Lef1 signaling regulates epaxial myogenesis via Pitx2 but that this link is uncoupled in other regions of the body, emphasizing the unique molecular networks that control the development of various muscles in vertebrates

Stress-Induced C/EBP Homology Protein (CHOP) Represses MyoD Transcription to Delay Myoblast Differentiation

When mouse myoblasts or satellite cells differentiate in culture, the expression of myogenic regulatory factor, MyoD, is downregulated in a subset of cells that do not differentiate. The mechanism involved in the repression of MyoD expression remains largely unknown. Here we report that a stress-response pathway repressing MyoD transcription is transiently activated in mouse-derived C2C12 myoblasts growing under differentiation-promoting conditions. We show that phosphorylation of the α subunit of the translation initiation factor 2 (eIF2α) is followed by expression of C/EBP homology protein (CHOP) in some myoblasts. ShRNA-driven knockdown of CHOP expression caused earlier and more robust differentiation, whereas its constitutive expression delayed differentiation relative to wild type myoblasts. Cells expressing CHOP did not express the myogenic regulatory factors MyoD and myogenin. These results indicated that CHOP directly repressed the transcription of the MyoD gene. In support of this view, CHOP associated with upstream regulatory region of the MyoD gene and its activity reduced histone acetylation at the enhancer region of MyoD. CHOP interacted with histone deacetylase 1 (HDAC1) in cells. This protein complex may reduce histone acetylation when bound to MyoD regulatory regions. Overall, our results suggest that the activation of a stress pathway in myoblasts transiently downregulate the myogenic program

Grain in a Plasma in the Presence of External Electric Field: Kinetic Calculation of Effective Potential and Ionic Drag Force

Kinetic calculations of the effective grain potential are presented for the case of weakly-ionized plasma in the external electric field. The drag force associated with the ionic drift in the external field is found. It is shown that the absorption of electrons and ions by the grain can cause the change of the direction of the drag force.Представлено кiнетичнi розрахунки ефективного потенцiалу порошинки для випадку слабоiонiзованої плазми у зовнiшньому електричному полi. Знайдено силу опору, яка пов’язана з дрейфом iонiв у зовнiшньому полi. Показано, що поглинання електронiв та iонiв порошинкою може привести до змiни напрямку сили опору

Xenopus pitx3 target genes lhx1 and xnr5 are identified using a novel three-fluor flow cytometry–based analysis of promoter activation and repression

Background: Pitx3 plays a well understood role in directing development of lens, muscle fiber, and dopaminergic neurons; however, in Xenopus laevis, it may also play a role in early gastrulation and somitogenesis. Potential downstream targets of pitx3 possess multiple binding motifs that would not be readily accessible by conventional promoter analysis. Results: We isolated and characterized pitx3 target genes lhx1 and xnr5 using a novel three-fluor flow cytometry tool that was designed to dissect promoters with multiple binding sites for the same transcription factor. This approach was calibrated using a known pitx3 target gene, Tyrosine hydroxylase. Conclusions: We demonstrate how flow cytometry can be used to detect gene regulatory changes with exquisite precision on a cell-by-cell basis, and establish that in HEK293 cells, pitx3 directly activates lhx1 and represses xnr5

Microplate-based chromatin immunoprecipitation method, Matrix ChIP: a platform to study signaling of complex genomic events

The chromatin immunoprecipitation (ChIP) assay is a major tool in the study of genomic processes in vivo. This and other methods are revealing that control of gene expression, cell division and DNA repair involves multiple proteins and great number of their modifications. ChIP assay is traditionally done in test tubes limiting the ability to study signaling of the complex genomic events. To increase the throughput and to simplify the assay we have developed a microplate-based ChIP (Matrix ChIP) method, where all steps from immunoprecipitation to DNA purification are done in microplate wells without sample transfers. This platform has several important advantages over the tube-based assay including very simple sample handling, high throughput, improved sensitivity and reproducibility, and potential for automation. 96 ChIP measurements including PCR can be done by one researcher in one day. We illustrate the power of Matrix ChIP by parallel profiling 80 different chromatin and transcription time-course events along an inducible gene including transient recruitment of kinases

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

Biomedical Discovery Acceleration, with Applications to Craniofacial Development

The profusion of high-throughput instruments and the explosion of new results in the scientific literature, particularly in molecular biomedicine, is both a blessing and a curse to the bench researcher. Even knowledgeable and experienced scientists can benefit from computational tools that help navigate this vast and rapidly evolving terrain. In this paper, we describe a novel computational approach to this challenge, a knowledge-based system that combines reading, reasoning, and reporting methods to facilitate analysis of experimental data. Reading methods extract information from external resources, either by parsing structured data or using biomedical language processing to extract information from unstructured data, and track knowledge provenance. Reasoning methods enrich the knowledge that results from reading by, for example, noting two genes that are annotated to the same ontology term or database entry. Reasoning is also used to combine all sources into a knowledge network that represents the integration of all sorts of relationships between a pair of genes, and to calculate a combined reliability score. Reporting methods combine the knowledge network with a congruent network constructed from experimental data and visualize the combined network in a tool that facilitates the knowledge-based analysis of that data. An implementation of this approach, called the Hanalyzer, is demonstrated on a large-scale gene expression array dataset relevant to craniofacial development. The use of the tool was critical in the creation of hypotheses regarding the roles of four genes never previously characterized as involved in craniofacial development; each of these hypotheses was validated by further experimental work

Lying low but ready for action: the quiescent muscle satellite cell

International audienceThe muscle satellite cell is essential for skeletal muscle regeneration. It is located on the muscle fibre, under the basal lamina as a quiescent cell, which becomes activated after injury, when it leaves the fibre, proliferates, and either undergoes myogenesis to form new fibres or reconstitutes the satellite cell pool. In this review, we discuss the cellular environment of the quiescent cell, including the extracellular matrix, which constitutes its niche. Cell adhesion molecules and some signalling pathways reinforce its quiescent state, whereas other signals lead to activation. We discuss how the satellite cell is ready to respond with the appropriate receptors, but protects its quiescence by mechanisms that include immobilization of ligands by extracellular matrix components and synthesis of inhibitors for intracellular signalling pathways and for metalloproteinases that break down the matrix and promote ligand processing and receptor activation. The quiescent satellite cell is also well protected against toxins and oxidative stress. It has a low metabolic rate, as shown by few active mitochondria and anaerobic glycolysis. Different subpopulations of quiescent satellite cells can be distinguished on the basis of cell surface markers and stem cell-like properties. We discuss the latter in the context of the small proportion of satellite cells that express high levels of Pax7, or that are derived from cells that have never activated the Myf5 myogenic determination gene. However, many quiescent satellite cells transcribe Myf5, but do not enter myogenesis because of post-transcriptional regulation, which prevents Myf5 protein accumulation. Post-transcriptional regulation, through microRNA repression of a potential cell cycle activator, further illustrates how these cells are ready for action