Abstract
Hey2 gene mutations in both humans and mice have been associated with multiple cardiac defects. However, the currently reported localization of Hey2 in the ventricular compact zone cannot explain the wide variety of cardiac defects. Furthermore, it was reported that, in contrast to other organs, Notch doesn’t regulate Hey2 in the heart. To determine the expression pattern and the regulation of Hey2, we used novel methods including RNAscope and a Hey2CreERT2 knockin line to precisely determine the spatiotemporal expression pattern and level of Hey2 during cardiac development. We found that Hey2 is expressed in the endocardial cells of the atrioventricular canal and the outflow tract, as well as at the base of trabeculae, in addition to the reported expression in the ventricular compact myocardium. By disrupting several signaling pathways that regulate trabeculation and/or compaction, we found that, in contrast to previous reports, Notch signaling and Nrg1/ErbB2 regulate Hey2 expression level in myocardium and/or endocardium, but not its expression pattern: weak expression in trabecular myocardium and strong expression in compact myocardium. Instead, we found that FGF signaling regulates the expression pattern of Hey2 in the early myocardium, and regulates the expression level of Hey2 in a Notch1 dependent manner.
Highlights
Hey[2], together with Hey[1], HeyL and their presumptive Drosophila homologue, dHey, is a member of a subfamily of hairy-related basic helix-loop-helix transcription factors[1,2], that are implicated in cell fate determination and boundary formation[3]
We used the RNAscope, which can detect single mRNA molecules and allows for in situ hybridization (ISH) and immunofluorescence staining in the same samples[15], and a Hey2CreERT2 knockin mouse line[17], to determine the expression level and pattern of Hey[2] and found a broader expression pattern of Hey[2] than previously reported
We examined the Hey[2] expression pattern by staining with estrogen receptor (ESR) using E9.5 hearts from Hey2CreERT2 knockin embryos, and consistently Hey[2] was strongly expressed in the compact zone and in the endocardial cells of the atrioventricular canal (AVC) and outflow tract (OFT) (Fig. 1c,c2 and c3), and weakly expressed in the cardiomyocytes of trabecular zone and the ventricular endocardial cells (Fig. 1c)
Summary
Hey[2], together with Hey[1], HeyL and their presumptive Drosophila homologue, dHey, is a member of a subfamily of hairy-related basic helix-loop-helix (bHLH) transcription factors[1,2], that are implicated in cell fate determination and boundary formation[3]. Despite profound cardiac defects caused by Hey[2] mutation in both humans and mice, the current knowledge of the Hey[2] expression pattern—that Hey[2] is expressed in the compact zone of the myocardium—cannot fully explain this broad range of congenital defects. It is unclear how Hey[2] enrichment in the ventricular compact myocardium could contribute to atrioventricular morphogenesis and semilunar valvular morphogenesis. We further found that FGF2 regulates the expression level of Hey[2] in a Notch[1] dependent manner
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