ABSTRACT
Defects in cardiac neural crest lead to congenital heart disease through failure of cardiac outflow tract and ventricular septation. In this report, we demonstrate a previously unappreciated role for the transcription factor Ets1 in the regulation of cardiac neural crest development. When bred onto a C57BL/6 genetic background, Ets1(-/-) mice have a nearly complete perinatal lethality. Histologic examination of Ets1(-/-) embryos revealed a membranous ventricular septal defect and an abnormal nodule of cartilage within the heart. Lineage-tracing experiments in Ets1(-/-) mice demonstrated that cells of the neural crest lineage form this cartilage nodule and do not complete their migration to the proximal aspects of the outflow tract endocardial cushions, resulting in the failure of membranous interventricular septum formation. Given previous studies demonstrating that the MEK/ERK pathway directly regulates Ets1 activity, we cultured embryonic hearts in the presence of the MEK inhibitor U0126 and found that U0126 induced intra-cardiac cartilage formation, suggesting the involvement of a MEK/ERK/Ets1 pathway in blocking chondrocyte differentiation of cardiac neural crest. Taken together, these results demonstrate that Ets1 is required to direct the proper migration and differentiation of cardiac neural crest in the formation of the interventricular septum, and therefore could play a role in the etiology of human congenital heart disease.
Subject(s)
Cell Differentiation/physiology , Cell Movement/physiology , Heart/embryology , Neural Crest/cytology , Proto-Oncogene Protein c-ets-1/metabolism , Aggrecans/metabolism , Animals , Blotting, Western , Butadienes/pharmacology , Cartilage/embryology , Cartilage/metabolism , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Movement/genetics , Chondrocytes/cytology , Chondrocytes/metabolism , Enzyme Inhibitors/pharmacology , Fluorescent Antibody Technique , Heart/drug effects , Heart Defects, Congenital/genetics , In Situ Hybridization , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Neural Crest/embryology , Nitriles/pharmacology , Proto-Oncogene Protein c-ets-1/genetics , Reverse Transcriptase Polymerase Chain Reaction , SOX9 Transcription Factor/metabolism , SOXD Transcription Factors/metabolismABSTRACT
Development of the heart valves is a complex process that relies on the successful remodeling of endocardial cushions. This process is dependent on a number of transcriptional regulators, including GATA4 and its interacting partner FOG-2. We have previously shown that the endocardial cushions in FOG-2 deficient mice are hyperplastic and fail to remodel appropriately, suggesting a defect late in endocardial cushion development. To elucidate this defect, we examined the later steps in endocardial cushion development including mesenchymal cell proliferation, differentiation, and apoptosis. We also measured myocardialization and endothelial-to-mesenchymal transformation (EMT) using previously described in vitro assays. We found no difference in the ability of the endocardial cushions to undergo myocardialization or in the rates of mesenchymal cell proliferation, differentiation, or apoptosis in the FOG-2 deficient cushions when compared to wild-type controls. However, using a collagen gel invasion assay, we found a 78% increase in outflow tract cushion EMT and a 35% increase in atrioventricular cushion EMT in the FOG-2 deficient mice when compared with wild-type mice. Taken together with GATA4's known role in promoting EMT, these results suggest that FOG-2 functions in cardiac valve formation as an attenuator of EMT by repressing GATA4 activity within the developing endocardial cushions.
Subject(s)
Cell Differentiation/genetics , DNA-Binding Proteins/genetics , Endocardium/embryology , Heart/embryology , Transcription Factors/genetics , Animals , Apoptosis/physiology , Cell Proliferation , DNA Primers , Endocardium/cytology , Endothelial Cells/cytology , GATA4 Transcription Factor/metabolism , In Situ Hybridization , Mesoderm/cytology , Mice , Mice, Knockout , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Eph receptor tyrosine kinases and ephrins are required for axon patterning and plasticity in the developing nervous system. Typically, Eph-ephrin interactions promote inhibitory events; for example, prohibiting the entry of neural cells into certain embryonic territories. Here, we show that distinct subsets of motor neurons that express EphA4 respond differently to ephrin-A5. EphA4-positive LMC(l) axons avoid entering ephrin-A5-positive hindlimb mesoderm. In contrast, EphA4-positive MMC(m) axons extend through ephrin-A5-positive rostral half-sclerotome. Blocking EphA4 activation in MMC(m) neurons or expanding the domain of ephrin-A5 expression in the somite results in the aberrant growth of MMC(m) axons into the caudal half-sclerotome. Moreover, premature expression of EphA4 in MMC(m) neurons leads to a portion of their axons growing into novel ephrin-A5-positive territories. Together, these results indicate that EphA4-ephrin-A5 signaling acts in a positive manner to constrain MMC(m) axons to the rostral half-sclerotome. Furthermore, we show that Eph activation localizes to distinct subcellular compartments of LMC(l) and MMC(m) neurons, consistent with distinct EphA4 signaling cascades in these neuronal subpopulations.
