ABSTRACT
Skeletal muscle is composed of heterogeneous populations of myofibers that are classified as slow- and fast-twitch fibers. The muscle fiber-type is regulated in a coordinated fashion by multiple genes, including transcriptional factors and microRNAs (miRNAs). However, players involved in this regulation are not fully elucidated. One of the members of the Vestigial-like factors, Vgll2, is thought to play a pivotal role in TEA domain (TEAD) transcription factor-mediated muscle-specific gene expression because of its restricted expression in skeletal muscles of adult mice. Here, we generated Vgll2 null mice and investigated Vgll2 function in adult skeletal muscles. These mice presented an increased number of fast-twitch type IIb fibers and exhibited a down-regulation of slow type I myosin heavy chain (MyHC) gene, Myh7, which resulted in exercise intolerance. In accordance with the decrease in Myh7, down-regulation of miR-208b, encoded within Myh7 gene and up-regulation of targets of miR-208b, Sox6, Sp3, and Purß, were observed in Vgll2 deficient mice. Moreover, we detected the physical interaction between Vgll2 and TEAD1/4 in neonatal skeletal muscles. These results suggest that Vgll2 may be both directly and indirectly involved in the programing of slow muscle fibers through the formation of the Vgll2-TEAD complex.
Subject(s)
Muscle Fibers, Skeletal/metabolism , Muscle Proteins/genetics , Muscle Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Animals , Gene Expression , Gene Expression Regulation , Genetic Loci , Mice , Mice, Knockout , MicroRNAs/genetics , Muscle Fibers, Fast-Twitch/metabolism , Muscle Fibers, Slow-Twitch/metabolism , Protein Binding , RNA, Messenger/geneticsABSTRACT
Embryonic stem cells are considered to be a good in vitro tool to study the induction of various cell types including cardiomyocytes; however, induction of the pharyngeal endoderm (PE), the underlying heart-forming region, in vivo has been scarcely reported. In the present study, we found that many PE-related genes, such as Paxl, Pax9, Sixl, and Tbxl, were up-regulated in cardiomyocyte-rich embryoid bodies (EBs). The third pouch-related genes including Hoxa3, Foxn1, and Aire, which are crucial for thymus development and function, were also detected in later stages. Nkx2.5, a cardiac transcription factor gene, is known to be transiently expressed in the PE. By crossing Nkx2.5-Cre mice with Cre-dependent EGFP reporter mice, we found that Nkx2.5(+) lineage exclusively contributed to thymic epithelial cell development, followed by thymus development. Gene expression analysis using Nkx2.5-EGFP ES cells also revealed that PE-related mRNAs were specifically enriched in the transiently appearing E-cadherin(+)Nkx2.5(+) cell fraction. Interestingly, the EB-derived cells were found capable of supporting T-cell differentiation to CD4 or CD8 double-positive cells in a reaggregation organ culture in vitro. Our results suggest that EBs contain cells that resemble third pharyngeal pouch endoderm and confer a thymus-like microenvironment.
