SUMMARY We created a whole-mount in situ hybridization (WISH) database, termed EMBRYS, containing expression data of 1520 transcription factors and cofactors expressed in E9.5, E10.5, and E11.5 mouse embryos—a highly dynamic stage of skeletal myogenesis. This approach implicated 43 genes in regulation of embryonic myogenesis, including a transcriptional repressor, the zinc-finger protein RP58 (also known as Zfp238). Knockout and knockdown approaches confirmed an essential role for RP58 in skeletal myogenesis. Cell-based high-throughput transfection screening revealed that RP58 is a direct MyoD target. Microarray analysis identified two inhibitors of skeletal myogenesis, Id2 and Id3, as targets for RP58-mediated repression. Consistently, MyoD-dependent activation of the myogenic program is impaired in RP58 null fibroblasts and downregulation of Id2 and Id3 rescues MyoD’s ability to promote myogenesis in these cells. Our combined, multi-system approach reveals a MyoD-activated regulatory loop relying on RP58-mediated repression of muscle regulatory factor (MRF) inhibitors.

Tightly regulated Ca2+ homeostasis is a prerequisite for proper cardiac function. To dissect the regulatory network of cardiac Ca2+ handling, we performed a chemical suppressor screen on zebrafish tremblor embryos, which suffer from Ca2+ extrusion defects. Efsevin was identified based on its potent activity to restore coordinated contractions in tremblor. We show that efsevin binds to VDAC2, potentiates mitochondrial Ca2+ uptake and accelerates the transfer of Ca2+ from intracellular stores into mitochondria. In cardiomyocytes, efsevin restricts the temporal and spatial boundaries of Ca2+ sparks and thereby inhibits Ca2+ overload-induced erratic Ca2+ waves and irregular contractions. We further show that overexpression of VDAC2 recapitulates the suppressive effect of efsevin on tremblor embryos whereas VDAC2 deficiency attenuates efsevin's rescue effect and that VDAC2 functions synergistically with MCU to suppress cardiac fibrillation in tremblor. Together, these findings demonstrate a critical modulatory role for VDAC2-dependent mitochondrial Ca2+ uptake in the regulation of cardiac rhythmicity.DOI: http://dx.doi.org/10.7554/eLife.04801.001

Glucocorticoids, such as dexamethasone (Dex), have been used as in vitro inducers of adipogenesis. However, the roles of the glucocorticoid receptor (GR) in adipogenesis have not been well characterized yet. Here we show that inhibition of GR activity using the GR antagonist RU486 prevents human mesenchymal stem cell (hMSC) and mouse embryonic fibroblast (MEF) differentiation into adipocytes. Moreover, in MEFs isolated from GR knockout (GRnull) and GRdim mice deficient in GR DNA-binding activity, adipogenesis was blocked. We identified GRE sites in the first intron of KLF15 by bioinformatical promoter analysis and confirmed their functional relevance by demonstrating GR interaction by chromatin immunoprecipitation. Moreover transfection of MEFs with siRNA for KLF15 significantly attenuated the expressions of adipogenic-marker genes and the lipid accumulation. Our results provide a new mechanism for understanding glucocorticoids dependent adipogenesis and that GR promotes adipogenesis via KLF15 gene expression as a transcriptional direct target.

Limb skeletal elements develop from a cartilage template, which is formed by the process termed chondrogenesis. This process is crucial in determining the shape and size of definitive bones in vertebrates. During chondrogenesis, aggregated mesenchymal cells undergo a highly organized process of proliferation and maturation along with secretion of extracellular matrix followed by programmed cell death and replacement by bone. The molecular mechanisms underlying this sophisticated process have been extensively studied. It has been demonstrated that several transcription factors such as Sox genes and Runx genes are indispensable for the major steps in chondrogenesis. Additionally, a number of signaling molecules including Bmps, Fgfs and Ihh/PTHrP are known to regulate chondrogenesis through highly coordinated interactions. This review is meant to provide an overview of the current knowledge of chondrogenesis with particular emphasis on the cellular and molecular aspects.

Objective Fluid shear stress intimately regulates vasculogenesis and endothelial homeostasis. The canonical Wnt/β-catenin signaling pathways play an important role in differentiation and proliferation. In this study, we investigated whether shear stress activated Angiopoietin-2 (Ang-2) via the canonical Wnt signaling pathway with an implication in vascular endothelial repair. Approach and Results Oscillatory shear stress(OSS) up-regulated both TOPflash Wnt reporter activities and the expression of Ang-2 RNA and protein in human aortic endothelial cells (HAEC) accompanied by an increase in nuclear β-catenin intensity. OSS-induced Ang-2 and Axin-2 mRNA expression was down-regulated in the presence of a Wnt inhibitor, IWR-1, but was up-regulated in the presence of a Wnt agonist, LiCl. Ang-2 expression was further down-regulated in response to a Wnt signaling inhibitor, DKK-1, but was up-regulated by Wnt agonist Wnt3a. Both DKK-1 and Ang-2 siRNA inhibited endothelial cell migration and tube formation, which were rescued by human recombinant Ang-2. Both Ang-2 and Axin-2 mRNA down-regulation was recapitulated in the heat-shock inducible transgenic Tg (hsp70l:dkk1-GFP) zebrafish embryos at 72 hours post fertilization (hpf). Ang-2 morpholino injection of Tg (kdrl:GFP) fish impaired subintestinal vessel (SIV) formation at 72hpf, which was rescued by zebrafish Ang-2 mRNA (zAng-2) co-injection. Inhibition of Wnt signaling with IWR-1 also down-regulated Ang-2 and Axin-2 expression, and impaired vascular repair after tail amputation, which was rescued by zAng-2 injection. Conclusion Shear stress activated Ang-2 via canonical Wnt signaling in vascular endothelial cells, and Wnt-Ang-2 signaling is recapitulated in zebrafish embryos with a translational implication in vascular development and repair.