Subject(s)
Carcinoma, Non-Small-Cell Lung/diagnosis , Central Nervous System Diseases/diagnosis , Facial Paralysis/etiology , Lung Neoplasms/diagnosis , Meningeal Carcinomatosis/diagnosis , Brain/diagnostic imaging , Carcinoma, Non-Small-Cell Lung/pathology , Central Nervous System Diseases/complications , Central Nervous System Diseases/diagnostic imaging , Diagnosis, Differential , Humans , Lung Neoplasms/pathology , Magnetic Resonance Imaging , Male , Meningeal Carcinomatosis/complications , Meningeal Carcinomatosis/secondary , Middle Aged , Missed Diagnosis , Neuroimaging , Sarcoidosis/complications , Sarcoidosis/diagnosis , Sarcoidosis/diagnostic imagingABSTRACT
The Nkx2-5 gene codes for a transcription factor that plays a critical role in heart development. Heterozygous mutations in NKX2-5 in both human and mice result in congenital heart defects (CHDs). However, the molecular mechanisms by which these mutations cause the disease are still unknown. Recently, we have generated the heterozygous mouse model of the human CHDs associated mutation NKX2-5 R142C (Nkx2-5R141C/+ mouse ortholog of human NKX2-5 R142C variant) that developed septal and conduction defects. This study generated a heterozygous Nkx2-5 R141C mouse embryonic stem cell line (Nkx2-5R141C/+ mESCs) to model CHDs in vitro. We observed that Nkx2-5R141C/+ mESCs display an alteration in the expression of genes that are essential for normal heart development. Furthermore, the reduced cardiomyogenesis is paralleled by a reduction in nuclear import of Nkx2-5 protein. Examination of the Nkx2-5R141C/+ embryos at E8.5 revealed a transient loss of cardiomyogenesis, which is consistent with the phenotype observed in vitro. Moreover, gene expression profiling of Nkx2-5R141C/+ cells at an early stage of cardiac differentiation revealed pronounced deregulation of several cardiac differentiation and function genes. Collectively, our data showed that heterozygosity for the R141C mutation results in disruption of the cellular distribution of Nkx2-5 protein, a transient reduction in cardiomyogenesis that may disrupt the early patterning of the heart, and this, in turn, affects the intricate orchestration of signaling pathways leading to downregulation of Bone morphogenetic protein (BMP) and Notch signaling. Therefore, we have developed mESCs model of a human CHD, providing an in vitro system to examine early stages of heart development, which are otherwise difficult to study in vivo. Stem Cells 2018;36:514-526.
Subject(s)
Bone Morphogenetic Proteins/metabolism , Heart Defects, Congenital/metabolism , Homeobox Protein Nkx-2.5/metabolism , Models, Cardiovascular , Mouse Embryonic Stem Cells/metabolism , Receptors, Notch/metabolism , Signal Transduction , Amino Acid Substitution , Animals , Bone Morphogenetic Proteins/genetics , Heart Defects, Congenital/genetics , Heart Defects, Congenital/pathology , Homeobox Protein Nkx-2.5/genetics , Humans , Mice , Mouse Embryonic Stem Cells/pathology , Mutation, Missense , Receptors, Notch/geneticsABSTRACT
Quiescent satellite cells are myogenic progenitors that enable regeneration of skeletal muscle. One of the early events of satellite cell activation following myotrauma is the induction of the myogenic regulatory factor MyoD, which eventually induces terminal differentiation and muscle function gene expression. The purpose of this study was to elucidate the mechanism by which MyoD is induced during activation of satellite cells in mouse muscle undergoing regeneration. We show that Six1, a transcription factor essential for embryonic myogenesis, also regulates MyoD expression in muscle progenitor cells. Six1 knock-down by RNA interference leads to decreased expression of MyoD in myoblasts. Chromatin immunoprecipitation assays reveal that Six1 binds the Core Enhancer Region of MyoD. Further, transcriptional reporter assays demonstrate that Core Enhancer Region reporter gene activity in myoblasts and in regenerating muscle depends on the expression of Six1 and on Six1 binding sites. Finally, we provide evidence indicating that Six1 is required for the proper chromatin structure at the Core Enhancer Region, as well as for MyoD binding at its own enhancer. Together, our results reveal that MyoD expression in satellite cells depends on Six1, supporting the idea that Six1 plays an important role in adult myogenesis, in addition to its role in embryonic muscle formation.
