Summary Identification of tissue-specific and developmentally active enhancers provides insights into mechanisms that control gene expression during embryogenesis. However, robust detection of these regulatory elements remains challenging, especially in vertebrate genomes. Here, we apply fluorescent activated nuclei sorting (FANS) followed by Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-Seq) to identify developmentally active endothelial enhancers in the zebrafish genome. ATAC-Seq of nuclei from Tg(fli1a:egfp)y1 transgenic embryos revealed expected patterns of nucleosomal positioning at transcriptional start sites throughout the genome and association with active histone modifications. Comparison of ATAC-Seq from GFP-positive and -negative nuclei identified more than 5,000 open elements specific to endothelial cells. These elements flanked genes functionally important for vascular development and that displayed endothelial-specific gene expression. Importantly, a majority of tested elements drove endothelial gene expression in zebrafish embryos. Thus, FANS-assisted ATAC-Seq using transgenic zebrafish embryos provides a robust approach for genome-wide identification of active tissue-specific enhancer elements.

Phosphoinositide 3-OH kinase (PI3K) regulates a number of developmental and physiologic processes in skeletal muscle; however, the contributions of individual PI3K p110 catalytic subunits to these processes are not well-defined. To address this question, we investigated the role of the 110-kDa PI3K catalytic subunit ␤ (p110␤) in myogenesis and metabolism. In C2C12 cells, pharmacological inhibition of p110␤ delayed differentiation. We next generated mice with conditional deletion of p110␤ in skeletal muscle (p110␤ muscle knockout [p110␤-mKO] mice). While young p110␤-mKO mice possessed a lower quadriceps mass and exhibited less strength than control littermates, no differences in muscle mass or strength were observed between genotypes in old mice. However, old p110␤-mKO mice were less glucose tolerant than old control mice. Overexpression of p110␤ accelerated differentiation in C2C12 cells and primary human myoblasts through an Akt-dependent mechanism, while expression of kinase-inactive p110␤ had the opposite effect. p110␤ overexpression was unable to promote myoblast differentiation under conditions of p110␣ inhibition, but expression of p110␣ was able to promote differentiation under conditions of p110␤ inhibition. These findings reveal a role for p110␤ during myogenesis and demonstrate that long-term reduction of skeletal muscle p110␤ impairs whole-body glucose tolerance without affecting skeletal muscle size or strength in old mice.

Skeletal muscle physiology and metabolism are regulated by complex networks of intracellular signaling pathways. Among many of these pathways, the protein kinase AKT plays a prominent role. While three AKT isoforms have been identified (AKT1, AKT2, and AKT3), surprisingly little is known regarding isoform‐specific expression of AKT in human skeletal muscle. To address this, we examined the expressions of each AKT isoform in muscle biopsy samples collected from the vastus lateralis of healthy male adults at rest. In muscle, AKT2 was the most highly expressed AKT transcript, exhibiting a 15.4‐fold increase over AKT1 and AKT3 transcripts. Next, the abundance of AKT protein isoforms was determined using antibody immunoprecipitation followed by Liquid Chromatography‐Parallel Reaction Monitoring/Mass Spectrometry. Immunoprecipitation was performed using either mouse or rabbit pan AKT antibodies that were immunoreactive with all three AKT isoforms. We found that AKT2 was the most abundant AKT isoform in human skeletal muscle (4.2‐fold greater than AKT1 using the rabbit antibody and 1.6‐fold greater than AKT1 using the mouse antibody). AKT3 was virtually undetectable. Next, cultured primary human myoblasts were virally‐transduced with cDNAs encoding either wild‐type (WT) or kinase‐inactive AKT1 (AKT1‐K179M) or AKT2 (AKT2‐K181M) and allowed to terminally differentiate. Myotubes expressing WT‐AKT1 or WT‐AKT2 showed enhanced fusion compared to control myotubes, while myotubes expressing AKT1‐K179M showed a 14% reduction in fusion. Myotubes expressing AKT2‐K181M displayed 63% decreased fusion compared to control. Together, these data identify AKT2 as the most highly‐expressed AKT isoform in human skeletal muscle and as the principal AKT isoform regulating human myoblast differentiation.

During early skeletal muscle regeneration in mice, transcript expressions for some components of the IGF-I/PI3K pathway are sensitive to initial injury intensity induced by freeze damage.