Abstract
The atrioventricular (AV) junction plays a critical role in chamber septation and transmission of cardiac conduction pulses. It consists of structures that develop from embryonic dorsal mesenchymal protrusion (DMP) and the embryonic AV canal. Despite extensive studies on AV junction development, the genetic regulation of DMP development remains poorly understood. In this study we present evidence that Shox2 is expressed in the developing DMP. Intriguingly, this Shox2-expressing domain possesses a pacemaker-specific genetic profile including Hcn4 and Tbx3. This genetic profile leads to nodal-like electrophysiological properties, which is gradually silenced as the AV node becomes matured. Phenotypic analyses of Shox2(-/-) mice revealed a hypoplastic and defectively differentiated DMP, likely attributed to increased apoptosis, accompanied by dramatically reduced expression of Bmp4 and Hcn4, ectopic activation of Cx40, and an aberrant pattern of action potentials. Interestingly, conditional deletion of Bmp4 or inhibition of BMP signaling by overexpression of Noggin using a Shox2-Cre allele led to a similar DMP hypoplasia and down-regulation of Hcn4, whereas activation of a transgenic Bmp4 allele in Shox2(-/-) background attenuated DMP defects. Moreover, the lack of Hcn4 expression in the DMP of mice carrying Smad4 conditional deletion and direct binding of pSmad1/5/8 to the Hcn4 regulatory region further confirm the Shox2-BMP genetic cascade in the regulation of DMP development. Our results reveal that Shox2 regulates DMP fate and development by controlling BMP signaling through the Smad-dependent pathway to drive tissue growth and to induce Hcn4 expression and suggest a temporal pacemaking function for the DMP during early cardiogenesis.
Highlights
Dorsal mesenchymal protrusion (DMP) is required for cardiac septation, but its additional functions are unknown
Shox2 expression in the sinoatrial node (SAN), venous valves, and sinus horns and other developing organs was confirmed by the LacZ reporter allele, we identified a novel Shox2 expression site that was not reported previously; that is, the developing AV junction from embryonic day 11.5 (E11.5) up to E18.5, at which time the expression became down-regulated as compared with persistent strong expression in the SAN (Fig. 1, A–C) [16]
Because the AV junction myocardium consists of dorsal mesenchymal protrusion (DMP) and atrioventricular node (AVN), we set out to determine the identity of this Shox2-expressing tissue by cell lineage tracing using the Shox2-Cre allele compounded with the R26R-LacZ reporter and performed an immunohistochemistry assay on the expression of pacemaker markers hyperpolarization-activated, cyclic nucleotide-gated 4 (Hcn4) and Tbx3 as well as myocardium marker Cx40
Summary
Dorsal mesenchymal protrusion (DMP) is required for cardiac septation, but its additional functions are unknown. The AV junction that lies at the merging point of cardiac chambers is critically involved in both septum and CCS developmental processes [2] It comprises the dorsally positioned second heart field-derived dorsal mesenchymal protrusion (DMP) that gives rise to the future vestibular spine [2] and ventrally positioned endocardial cushion tissue as well as AV canal-derived myocardium including AVN precursors [3, 4], The outgrowth and fusion of DMP and endocardial cushion tissue are essential for proper formation of AV septation [2, 5]. In this study we identified Shox expression, overlapping with that of pacemaker-specific genes including Hcn and Tbx (24 –26), in the early developing DMP that manifests a nodal-like electrophysiological character, suggesting a potential pacemaking function for the embryonic DMP during cardiogenesis.
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