Segregation of Central Ventricular Conduction System Lineages in Early SMA+ Cardiomyocytes Occurs Prior to Heart Tube Formation

The cardiac conduction system (CCS) transmits electrical activity from the atria to the ventricles to coordinate heartbeats. Atrioventricular conduction diseases are often associated with defects in the central ventricular conduction system comprising the atrioventricular bundle (AVB) and right and left branches (BBs). Conducting and contractile working myocytes share common cardiomyogenic progenitors, however the time at which the CCS lineage becomes specified is unclear. In order to study the fate and the contribution to the CCS of cardiomyocytes during early heart tube formation, we performed a genetic lineage analysis using a Sma-CreERT2 mouse line. Lineage tracing experiments reveal a sequential contribution of early Sma expressing cardiomyocytes to different cardiac compartments, labeling at embryonic day (E) 7.5 giving rise to the interventricular septum and apical left ventricular myocardium. Early Sma expressing cardiomyocytes contribute to the AVB, BBs and left ventricular Purkinje fibers. Clonal analysis using the R26-confetti reporter mouse crossed with Sma-CreERT2 demonstrates that early Sma expressing cardiomyocytes include cells exclusively fated to give rise to the AVB. In contrast, lineage segregation is still ongoing for the BBs at E7.5. Overall this study highlights the early segregation of the central ventricular conduction system lineage within cardiomyocytes at the onset of heart tube formation.


Introduction
The cardiac conduction system (CCS) generates and propagates electrical impulses to coordinate atrial and ventricular contraction. The pacemaker or sinoatrial node (SAN) initiates electrical activity that transits through the atrioventricular node (AVN), the unique electrical pathway between atria and ventricles. Subsequently, the ventricular conduction system (VCS), comprised of central components including the atrioventricular or His-Bundle (AVB) and bundle branches (BB) and the peripheral Purkinje Fiber (PF) network, leads to the rapid propagation of electrical activity to trigger apex to base ventricular contraction [1]. Defects in the propagation of electrical activity are a leading cause of conduction defects and atrioventricular (AV) blocks [2]. Defining the developmental origin of the AV conduction pathway is an indispensable step towards understanding the etiology of conduction diseases.
In the mouse embryo, the first heartbeats are detected at embryonic day (E) 8.5 as peristaltic contractions of the primary cardiac tube, which are replaced by sequential contractions of the atria and
For lineage analysis, Sma-CreERT2 males were crossed with R26R females and 4-Hydroxytamoxifen (4-OHT) was injected intraperitoneally to pregnant female at different timepoints (E7.5 and E8.5). 4-OHT (Sigma, Saint-Louis, MO, USA, H7904) was dissolved at a concentration of 20 mg/mL in 100% ethanol, then diluted in Cremophor ® EL (Sigma) to 10 mg/mL. Before injection, 4-OHT was diluted in 1X PBS to 3 mg/mL and 200 µL of this solution was injected intraperitoneally into pregnant females. For prospective clonal analysis, Sma-CreERT2 males were crossed with R26-confetti females and the dose of 4-OHT injected was reduced to 100 to 400 µg per female.
For whole-mount immunofluorescence, embryonic hearts were dissected and fixed in 2% paraformaldehyde for 2-6 h at 4˝C, washed in PBS, permeabilized in PBS 1X/0.5% Triton X100 for 1 h and incubated for 3 h in saturation buffer (PBS 1X; 3% BSA; 0.1% Triton X100). The primary antibodies were incubated in saturation buffer for overnight at 4˝C. Secondary antibodies coupled to fluorescent molecules were incubated in saturation buffer and after washes, hearts were observed under a Zeiss Lumar stereomicroscope V12 or Zeiss LSM780 confocal microscope. For immunofluorescence on cryostat sections, we performed the same procedure as described previously [19].

Clonal Analysis
SMA-CreERT2 mice were crossed with Rosa-confetti reporter mice and low dose of 4-OH tamoxifen injections were performed at E7.5 to induce rare recombination events, generating well-separated clones. At P7, hearts were dissected and fixed and fluorescent clones were visualized under Zeiss Lumar stereomicroscope V12 using appropriate filters to detect RFP, GFP/YFP, and CFP. Hearts were then processed for cryosections and stained with an anti-Contactin-2 antibody as described above. Individual clusters were identified on serial sections and classified by color and localization. A single clone corresponds to a cluster composed of cells of only one color (RFP, GFP, YFP, or CFP) that is distributed in consecutive sections. Clones with 100% or a restricted number of Cntn-2-positive cells were classified as conducting or mixed, respectively, while those with no Cntn-2-positive cells were classified as working.

SMA+ Cells in the Early Heart Tube Contribute to the Interventricular Septum
Using whole-mount immunofluorescence, SMA expression was detected at E7.5 in the anterior medioventral position of the cardiac crescent expressing the transcription factor Nkx2-5 ( Figure 1A). At E8.5, SMA is uniformly present in the primary heart tube while Nkx2-5 expression is also observed in the sinus venosus region ( Figure 1C). SMA+ early cardiomyocytes thus appear to identify differentiated myocardial cells within the primary heart tube. To establish the fate of these early cardiomyocytes during heart development we performed a genetic tracing of SMA+ cells. Sma CreER2{`m ice were crossed with mice carrying a R262R LacZ{c onditional lineage reporter and Cre was activated upon 4-hydroxytamoxifen injections at early stages of development. After Cre activation at E7.5 or E8.5, β-galactosidase expression was observed only in the heart of E11.5 embryos ( Figure 1B,D), highlighting the cardiac restricted expression of SMA at early developmental stages. Closer examination of these hearts revealed different β-galactosidase staining patterns between these two timepoints. Cre induction at E7.5 results in labeling of the interventricular region and part of the left ventricle, both derivatives of the FHF, while both atria, the outflow tract (OFT), the right ventricle, and atrioventricular canal are devoid of β-gal+ cells. (Figure 1B',B"). No labeling was observed in the outer curvature of the left ventricle (arrow in Figure 1B'). Cre induction at E8.5, in contrast, results in β-gal+ cells in all cardiac compartments with the exception of the outflow tract and the sinus venosus ( Figure 1D',D"), two regions derived from the SHF, and the last cells to enter the heart tube [23]. These results highlight the progressive activation of Sma as different compartments of the heart form, revealing consecutive formation of the interventricular region and the left ventricle, then the atria, the right ventricle and the atrioventricular canal and finally the OFT and SV.
The contribution of early SMA+ cells induced at E7.5 was investigated at later stages of heart development. At E12.5, a comparable staining with that at E11.5 ( Figure 1B',B") was observed ( Figure 1E,E'). Sections of these hearts show β-gal staining in the interventricular septum (IVS) and both compact and trabecular zones of the LV ( Figure 1F,F'). SMA-derived cells are found in the entire IVS, however the upper part of the septum contained fewer and more sparse β-gal+ cells compared to the apical part of the IVS. At later stages, these SMA+ cardiac progenitors contribute predominantly to the formation of the IVS and trabeculae of the LV; only very few β-gal+ cells are observed in the compact myocardium in the apex of the LV ( Figure 1G,H). Together, these data show that SMA+ cardiac cells at E7.5 form the primary heart tube and contribute at later stages to the formation of the interventricular septum and left ventricular trabeculae, consistent with FHF contributions to the developing heart.

SMA+ Early Cardiac Cells Contribute to the Ventricular Conduction System
In order to investigate the cell lineage contribution of these SMA+ early cardiac cells to the ventricular conduction system (VCS), R262R LacZ{`: :Sma CreER2{`m ice were crossed with Cx40 GFP{m ice expressing GFP specifically in the VCS at fetal and adult stages [19]. Cre activation was performed by tamoxifen injection at E7.5. Hearts recovered at E18.5 or P7 were dissected to isolate the IVS before X-gal staining. At E18.5, β-gal labeling was mainly detected on the left side of the septum (Figure 2A') and at the crest of the septum in the region of the AVB (arrow in Figure 2A). To further investigate the contribution of these cells to the VCS, we performed immunofluorescence on sections using anti-β-gal and Tbx5 antibodies. Co-expression of Cx40-GFP or Tbx5 with β-gal is observed in both bundle branches (BB) and the AVB ( Figure 2B,B',C,C'). Only very few β-gal+ cells were detected in the AVN ( Figure 2D), which is known to derive from the AVC [24]. These results were confirmed in postnatal hearts (P7), by which stage the definitive VCS is established [25]. Extensive labeling was observed on both sides of the IVS ( Figure 2E,F) and β-gal+ cells were observed along the crest of the IVS and both sides of the septum ( Figure 2E',F'). This distribution of β-gal+ cells is characteristic of the pattern of the VCS. To confirm that early SMA+ cardiomyocytes contribute to the VCS, immunofluorescence with Contactin-2 and β-gal antibodies was performed and revealed double positive cells in the AVB, BB, and left Purkinje Fibers ( Figure 2G,H). These results demonstrate that early SMA+ cardiomyocytes contribute to the formation of the VCS but not to the AVN.

The Central Ventricular Conduction System Lineage Segregates from SMA+ Early Cardiomyocytes
Working and conducting cardiomyocytes are derived from common progentior cells [25], although the time of working and VCS lineage divergence is unknown. In order to investigate whether E7.5 SMA+ cardiomyocytes giving rise the VCS have already segregated from cells giving rise to working myocardium, Sma CreER2{`m ice were crossed with R26R con f etti{`l ineage reporter mice to generate multicolor clones [15]. R26R-confetti mice possess four fluorescent reporter genes (RFP, CFP, YFP, and GFP) that are randomly activated upon Cre deletion [20]. We performed pulse activation of Cre recombinase by injecting 4-hydroxytamoxifen into pregnant females at E7.5. At E14.5, multicolor clusters were detected in the interventricular septum ( Figure 3A,B). Observations from a large number of hearts with multicolor clones revealed two types of clusters. In the apical two-thirds of the IVS, large and elongated unicolor clones are juxtaposed and distributed among the vertical apex to base axis ( Figure 3A,B). In contrast, in the upper part of the IVS, the distribution of labeled cells is more heterogenous with sparse randomly-distributed cells. To investigate the contribution of these labeled cells to the AVB and BB, Hcn4 immunofluorescence was performed on sections. Few labeled cells were found in the AVB and the clonal relationship between independent cells of the same color in the AVB was difficult to establish because of the dispersed distribution ( Figure 3C). In order to perform a clonal lineage analysis of the SMA+ early cardiomyocytes, we used a low dose of 4-hydroxytamoxifen to obtain between 1 and 20 multicolor clusters per heart. First, we observed the distribution of these clusters in whole-mount dissected IVS from postnatal day 7 (P7) hearts. Individual clusters of uniform color could be readily distinguished on the left side of the IVS (Figure 3D), therefore cells from each cluster are considered to be clonally related. Large and compact clones are found in the IVS while a RFP+ clone of sparse cells can be seen in the upper part of the septum where the AVB develops ( Figure 3D,D'). As all these sparse cells express the same color, they are considered to be part of the same clone. In total, we obtained a collection of 190 clusters from which 26 are in the central VCS defined by Contactin-2 immunostaining on sections. In the upper part of the IVS, three types of clones could be identified in relation to their contribution or not to the AV conduction system: no labeled cells in the AV conduction system (working, Figure 3E); few cells in the central VCS (Mixed, Figure 3E') or all cells in the central VCS (Conducting, Figure 3E"). In mixed clones, cells are distributed along both sides of the IVS (Figure 3E'). In contrast to working clones, cells in conducting ( Figure 3E") and mixed clones show a dispersed distribution, suggesting a different mode of growth compared to compact working clones. The number of exclusively conducting clones is twice the number of mixed clones ( Figure 3G). Finally, we studied the distribution of the mixed and conducting clones in different components of the central VCS ( Figure 3H). The nine clones identified in the AVB are entirely Contactin-2+ ( Figure 3F) while only half of the clones covering the BBs are exclusively conducting. Early Sma expressing cardiomyocytes therefore include cells exclusively fated to give rise to the AVB, suggesting early segregation of central VCS lineages. In the case of the BB component, lineage segregation is still ongoing at E7.5. This result is consistent with the existence of a dedicated population of central VCS precursors expressing Sma at the early heart tube stage.

SMA-Derived Early Cardiomyocytes form the Primary Heart Tube
To study the contribution of these early SMA+ cardiomyocytes to the primary heart tube, we performed a genetic tracing by crossing Sma CreER2{`m ice with R26R YFP{`. 4OH-tamoxifen was injected to pregnant females at E7.5 and embryos were recovered 20 h later at E8.5. YFP+ cells are detected in the central region of the heart tube but not at the cardiac poles ( Figure 4A-E). These data are consistent with the conclusion that the early heart tube gives rise predominantly to the IVS and only part of the LV.
To better understand where the precursors of the central VCS are localized in the early heart tube, we compared the expression of Nkx2-5, HCN4, and SMA in embryos at E8.0. Hcn4 expression is dynamic during cardiac development and temporally controlled lineage tracing of HCN4-derived cells reveals that their contribution to the AVB is low at E7.5 and high at E8.5 [14]. These results suggest that HCN4 expression is low in AVB precursors in the hours following tamoxifen injection at E7.5. At the 3-5 somites stage, SMA is detected in the heart tube while HCN4 expression is more intense around the sinus venosus and absent at the arterial pole ( Figure 4F,G). Immunofluorescence on sections shows co-labeling of SMA with Nkx2-5 in cardiomyocytes within the heart tube but not in progenitor cells at the arterial and venous poles ( Figure 4H). Taking into account the lineage tracing of SMA and HCN4 at E7.5 and E8.5, and their respective expression profiles, we conclude that AVB precursors localize in the anterior region of the heart tube at E8.0 ( Figure 4I).

Discussion and Conclusions
Cre-mediated genetic labeling has accelerated our understanding of how different cell lineages contribute to the heart. It is now well established that the primary heart tube forms the left ventricle while the other cardiac compartments are added during subsequent development at the venous and arterial poles [9]. This conclusion is based on the analysis of mouse mutants and genetic tracing showing that the LIM homeodomain transcription factor Isl1 is selectively expressed in SHF cells, which will form the outflow tract, right ventricle, right atria, and a portion of cells of the left ventricle and left atria. More recently, studies using inducible HCN4-CreERT2 and Tbx5-CreERT2 mice have shown that early progenitor cells labeled at E7.0 or E6.5, respectively, delineate FHF derivatives such as the left ventricle and both atria [13,14,26]. The results of our genetic Cre lineage analysis of early Sma+ cardiomyocytes are consistent with a progressive contribution of cells to the heart, with the earliest Sma+ cells giving rise to the interventricular septum. This conclusion is in agreement with data from early fate mapping of the cardiac tube in chicks showing that the primordium of the interventricular septum appears at the site of ventral fusion of bilateral cardiac primordia [27,28]. After Cre activation at E7.5, Sma+ cells give rise exclusively to the IVS and left ventricular apex. With Cre activation one day later, labeled cells were observed in all cardiac compartments, with the exception of the OFT region and sinus venosus which contribute later to the heart from the SHF. The boundary between the IVS and LV has been already documented using a Tbx2-Cre mouse line suggesting that the LV free wall myocardium derives from progressive addition of cells from the AVC [24]. The transcription factor Tbx2 is expressed early in the AVC and lineage tracing of these cells shows that they contribute to a large part of the left ventricular free wall, but not to the septum or LV apex. Similarly, Cre activation at gastrulation (E6.0) and early crescent stage (E7.0) of Hcn4+/FHF cells demonstrated a demarcation between labeling events in the IVS and the LV with the LV but not IVS being labeled when tamoxifen was injected at E7.0 [13]. It has been described that Hcn4 expression decreases as FHF progenitors are incorporated and that at the early heart tube stage, HCN4 positive cells are localized close to the venous pole [13]. Genetic tracing lineage analysis of Tbx5 labeled at E6.5 confirmed that early FHF cells give rise to the IVS and LV [26]. As the expression patterns of Hcn4 and Tbx5 are largely overlapping in the presumptive FHF [13], these data suggest that the IVS is derived from FHF progenitor cells that have already started to downregulate Hcn4 expression at the early heart tube stage. In contrast to Isl1, Hcn4, or Tbx2, SMA is stably expressed in early cardiomyocytes and is not restricted to specific cardiac compartments, so our data directly reflect the time of differentiation of these cells as they incorporate to the heart tube. Short-term lineage tracing of Sma+ cardiomyocytes shows that these cells contribute to the early heart tube but not to myocardium at the cardiac poles. In summary, the first Sma+ cardiomyocytes giving rise to the IVS are presumably negative for Tbx2 and Hcn4 and are localized in the anterior part of the early heart tube ( Figure 4I).
The contribution of FHF and SHF progenitors to the development of the central VCS was previously studied using several genetic approaches [10]. From these studies, FHF cells were shown to contribute exclusively to the LBB and Left PF while SHF cells contribute to the SAN and right Purkinje fibers. However, a dual contribution of cells from both heart fields has been proposed during the formation of the AVN, AVB, and RBB [12,14]. In our SMA genetic tracing experiments, we showed that the early cardiomyocytes contributing to the IVS also give rise to the AVB, LBB, and a small contribution to the RBB but almost none to the AVN. This result suggests that precursors of the central VCS are included in these early cardiomyocytes and that the formation of the definitive central VCS necessitates the contribution of cells from the AVC to form the AVN and SHF to form the AVB and RBB, as demonstrated by Tbx2 and Isl1 genetic tracing experiments [24,29]. Together, these data suggest that precursors of the VCS originate from both heart fields.
Previous studies have suggested that a population of early cardiomyocytes expressing GlN2 in human embryonic hearts localized at the inner curvature forms a primary ring at the interventricular foramen corresponding to the central VCS [5]. However, the lineage relationship between the embryonic primary ring and the definitive central VCS was unclear. Our clonal labeling of early cardiac cells highlights the early lineage segregation to the central VCS before formation of the primary ring. These data suggest that early cardiomyocytes are assigned to a conductive fate in the heart and maintain this function until postnatal stages. This is particularly true for the AVB and reinforces our earlier identification of a clonal boundary between the AVB and the upper septum [12]. However, in our previous retrospective clonal analysis, it was not possible to date this lineage segregation. The comparison of expression patterns between Hcn4 and SMA at E8.0 suggests that these precursors are included in the anterior part of the primary heart tube that gives rise to the IVS.
Using Hcn4CreERT2 mice, tamoxifen injection at E7.5 labeled FHF derivatives including the LBB but only partially labeled the AVB, which is strongly labeled after tamoxifen injection at E8.5, suggesting that the AVB differentiates after the LBB [14]. However, in our prospective clonal analysis of SMA+ early cardiomyocytes, we observe segregation of the AVB lineage at the early heart tube stage. The presence of mixed clones with cells in the conduction system and in the IVS suggests that the separation between these two lineages is not complete at this early stage of development for the right and left BB. These data are in accordance with our previous retrospective clonal analysis indicating the existence of early common progenitors for conductive and working cardiomyocytes [12,25]. Our new data using SMA prospective clonal analysis suggests that the AVB is specified earlier than the BB or during a more restricted time window. The discrepancy between our conclusions and those drawn from the Hcn4 fate mapping analysis is likely to result from the different expression patterns of these genes and the experimental approach. Indeed, the expression pattern of Hcn4 is particularly complex during cardiac development with an early expression in FHF progenitors and, from E16.5, a restriction to the CCS [13,14]. Moreover, clonal analysis is a strategy more appropriate to distinguish lineage segregation. Together, these data demonstrate that the segregation of the AVB lineage takes place as early as the heart tube stage in early Sma+ cardiomyocytes, in which re-expression or de novo expression of Hcn4 has not yet occurred ( Figure 4I). This conclusion suggests that segregation of the AVB lineage takes place slightly before the onset of the first cardiac contractions at the 5-somite stage [30]. Potentially, distinct AVB progenitor cells may exist even prior to myocardial differentiation. Overall, our data suggest a progressive segregation of the central VCS derived from early cardiomyocytes starting with the AVB, followed by cells forming the right and left BB. The presence of mixed clones shows that growth of the definitive central VCS requires the later addition of cells from other sources of progenitors. Indeed the pattern of these clones at the subendocardial layer along a vertical axis suggests an extension of the septum crest on both sides accompanying the growth of the IVS as has been described for the development of the BB [31]. It remains to be seen if lineage segregation of these cells occurs in the early heart or if adjacent cardiomyocytes contribute to the central VCS during later heart development.
Further understanding of the molecular and cellular basis of these lineage segregation events, and more precisely, the early formation of specialized conducting cardiomyocytes from common precursors with working myocytes will be of great relevance to conduction diseases and for regenerative strategies to replace the conduction system by bio-artificial pacemakers.