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

Implementing video conferencing in mental health practice

The aim of this paper is to provide an overview of the evidence base regarding the use of video conferencing (VC), implementation issues, policies, procedures, technical requirements and VC etiquette. The paper is based on a literature review of VC within the mental health sector and the authors' experience in implementing VC. Six themes emerged from the literature review: applications of VC, VC assessments, treatment, training and supervision, practitioner anxiety, and VC administrative processes. The results of the review support the use of VC in mental health services. Guidelines for the implementation of VC are discussed, including the importance of staff and service user consultations, training in the use of VC, clear guidance for staff with regards to usage, confidentiality and data protection policies, and VC etiquette. Challenges that can arise when implementing VC in a mental health context are also discussed. Arguably, it is not the technology, but the cultural change it represents to staff which seems to be the most important factor regarding successful implementation

Combinatorial signaling microenvironments for studying stem cell fate

Extracellular matrix (ECM) and growth factor signaling networks are known to interact in a complex manner. Therefore, reductionist approaches that test the cellular response to individual ECM components and growth factors cannot be used to predict the response to more complex mixtures without knowledge of the underlying signaling network. To address this challenge, we have developed a technology platform to experimentally probe the interactions of ECM components and soluble growth factors on stem cell fate. We present a multiwell microarray platform that allows 1200 simultaneous experiments on 240 unique signaling environments. Mixtures of extracellular matrix (fibronectin, laminin, collagen I, collagen III, collagen IV) are arrayed using a robotic spotter and arranged in a multiwell format. Embryonic stem (ES) cells adhere to ECM spots and are cultured in mixtures of soluble factors [wnt3a, activin A, bone morphogenetic protein-4 (BMP-4), and fibroblast growth factor-4 (FGF- 4)]. Differentiation along the cardiac lineage is monitored by myosin heavy chain--green fluorescent protein (MHC-GFP) reporter expression as compared to growth by monitoring nuclear DNA, and both signals are quantified using a confocal microarray scanner. In developing the platform, we characterized the amount of deposited protein, the fluorescent readout of GFP expression and DNA content, and the use of a laser-based scanner as compared to fluorescent microscopy for data acquisition. The effects of growth factors on growth and differentiation are consistent with previously reported literature, and preliminary evidence of interactive signaling is illuminated. This versatile technique is compatible with virtually any set of insoluble and soluble cues, leverages existing software and hardware, and represents a step toward developing the ‘systems biology’ of stem cells

Myocardin is a bifunctional switch for smooth versus skeletal muscle differentiation

Skeletal and smooth muscle can mutually transdifferentiate, but little molecular insight exists as to how each muscle program may be subverted to the other. The myogenic basic helix–loop–helix transcription factors MyoD and myogenin (Myog) direct the development of skeletal muscle and are thought to be dominant over the program of smooth muscle cell (SMC) differentiation. Myocardin (Myocd) is a serum response factor (SRF) coactivator that promotes SMC differentiation through transcriptional stimulation of SRF-dependent smooth muscle genes. Here we show by lineage-tracing studies that Myocd is expressed transiently in skeletal muscle progenitor cells of the somite, and a majority of skeletal muscle is derived from Myocd-expressing cell lineages. However, rather than activating skeletal muscle-specific gene expression, Myocd functions as a transcriptional repressor of Myog, inhibiting skeletal muscle differentiation while activating SMC-specific genes. This repressor function of Myocd is complex, involving histone deacetylase 5 silencing of the Myog promoter and Myocd's physical contact with MyoD, which undermines MyoD DNA binding and transcriptional synergy with MEF2. These results reveal a previously unrecognized role for Myocd in repressing the skeletal muscle differentiation program and suggest that this transcriptional coregulator acts as a bifunctional molecular switch for the smooth versus skeletal muscle phenotypes