HCN4 Dynamically Marks the First Heart Field and Conduction System Precursors

Abstract

Rationale To date, there has been no specific marker of the first heart field to facilitate understandings of contributions of the first heart field to cardiac lineages. Cardiac arrhythmia is a leading cause of death, often resulting from abnormalities in the cardiac conduction system (CCS). Understanding origins and identifying markers of CCS lineages is an essential step toward modeling diseases of the CCS and for development of biological pacemakers. Objective To investigate HCN4 as a marker for the first heart field and for precursors of distinct components of the CCS, and gain insight into contributions of first and second heart lineages to the CCS. Methods and Results HCN4-CreERT2, -nuclear LacZ and -H2BGFP mouse lines were generated. HCN4 expression was examined by means of immunostaining with HCN4 antibody and reporter gene expression. Lineage studies were performed using HCN4CreERT2, Isl1Cre, Nkx2.5Cre, and Tbx18Cre, coupled to co-immunostaining with CCS markers. Results demonstrated that, at cardiac crescent stages, HCN4 marks the first heart field, with HCN4CreERT2 allowing assessment of cell fates adopted by first heart field myocytes. Throughout embryonic development, HCN4 expression marked distinct CCS precursors at distinct stages, marking the entire CCS by late fetal stages. We also noted expression of HCN4 in distinct subsets of endothelium at specific developmental stages. Conclusions This study provides insight into contributions of first and second heart lineages to the CCS and highlights the potential utility of HCN4 in conjunction with other markers for optimization of protocols for generation and isolation of specific conduction system precursors.

A common Shox2-Nkx2-5 antagonistic mechanism primes the pacemaking cell fate in the pulmonary vein myocardium and sinoatrial node

“…S1D,E). The PV myocardium was believed to be derived from a lineage, distinct from that of the systemic venous return that exhibits characteristics similar to pacemaker cells in the developing embryo (Ammirabile et al, 2012;Liang et al, 2013;Mommersteeg et al, 2007a;Vedantham et al, 2013), but the colocalization of Shox2 with Hcn4 in the PV myocardium suggests a similar genetic pathway and origin for pacemaker fate in these two structures. Notably, Shox2 expression was strong in the myocardial cells surrounding the forming PV from E11.5 onwards (supplementary material Fig.…”

“…Subsequently, the SAN is identified as a structure comprising an Nkx2-5 − head region and an Nkx2-5 + sinoatrial (SA) junction or tail region (Liang et al, 2013;Liu et al, 2007;Wiese et al, 2009;Yamamoto et al, 2006), suggesting that the development of these two distinct SAN domains (Nkx2-5 + versus Nkx2-5 − ) is regulated by different mechanisms. The SAN is characterized by the expression of Hcn4, Tbx3, Isl1 and Shox2, but is negative for Cx40 (Gja5) and Nppa (Munshi, 2012).…”

“…Nkx2-5 was shown to be essential for maintaining the Cx40 + /Hcn4 − cell fate in the PV myocardium and for establishing a strict boundary of the SAN domain from the surrounding atrial working myocardium by inhibiting Hcn4 but activating Cx40 expression (Mommersteeg et al, 2007b). However, Nkx2-5 expression was also found in the SA junction region that is Hcn4 + /Cx40 − (Liang et al, 2013;Wiese et al, 2009), suggesting the existence of a mechanism that blocks the transcriptional output of Nkx2-5 (i.e. the transcription of Nkx2-5 target genes).…”

Properties of Cardiac Progenitor Cells in the Second Heart Field

“…This modeling approach highlights the temporal distinction between the FHF and SHF and can be used to predict gene function, providing an important step toward integrated understanding of regulatory networks during early heart development. The discovery that Hcn4, encoding a nucleotide-gated channel protein, is expressed in the FHF, in a complementary pattern to the SHF gene Isl1, has reinforced the concept that the vertebrate heart is built from distinct progenitor cell populations [4,5]. An inducible Cre allele of Hcn4 has allowed evaluation of the contribution of the FHF to the definitive heart.…”

“…Unlike the SHF, which contains multipotent cardiovascular progenitor cells, FHF derivatives appear to be restricted to myocardium [4]. Interestingly, the two lineages contribute differently and in a complementary manner to different components of the cardiac conduction system [5]. In particular, the right bundle branch and majority of the right Purkinje fiber system have a SHF origin.…”