Disposition of CCS in Cryptic–/– embryos at E18.5. To investigate CCS disposition in mouse heterotaxy, we first examined the hearts of Cryptic–/– embryos at E18.5, which develop right isomerism (32, 33). Although cardiac looping is reported to be randomized in Cryptic–/– mice (32), embryos carrying the same mutation on an FVB/N background showed a d-loop (n = 7/7 at E18.5; n = 8/8 at E14.5; n = 6/6 at E12.5), indicating that the looping phenotype depends on the genetic background. Characteristic congenital heart diseases (CHDs) included common atrium (n = 7/7), atrioventricular septal defect (AVSD) (n = 7/7), transposition of the great arteries (TGA) (n = 5/7), double outlet right ventricle (DORV) (n = 2/7), and total anomalous pulmonary venous connection (TAPVC) (n = 7/7) — features typically observed in human right atrial isomerism (34) (see Supplemental Table 1 for full description of mutant hearts; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.199072DS1).

Specialized cardiomyocytes were detected using in situ hybridization of Hcn4, followed by 3D reconstruction of the CCS (WT, n = 6; Cryptic–/–, n = 4) (Figure 1, A–L). In Cryptic–/– embryos, bilateral SA nodes were present, albeit with smaller sizes compared with that in the WT embryos (n = 4/4) (Figure 1, B and H). Although the AV node was normally connected to the AV bundle, its position had shifted caudally (Figure 1, A and F). Left and right bundle branches were identified as continuations of the AV bundle, retaining their characteristic morphologies (Supplemental Figure 1). Notably, a distinct, intensively Hcn4-expressing nodule was detected in the ventricle, continuous with the AV ring at the cranioventral position (n = 4/4) (Figure 1, H and L). Immunostaining confirmed the presence of Hcn4 protein in the SA nodes and cranioventral nodule of the Cryptic–/– heart, consistent with the in situ hybridization results (Supplemental Figure 2). In humans, right atrial isomerism is accompanied by bilateral SA nodes and dual AV nodes that often connect with the AV bundles to form the sling (34, 35, 37). To confirm the identity of the Hcn4+ nodule as an anterior AV node, we examined additional markers — Tbx3 and Myl7 for the AV node (12, 38) and Tbx3 and Gja5 for the AV bundle (12, 41) — in 3 Cryptic–/– hearts. The nodule was Tbx3+/Myl7+ (n = 3/3) (Figure 1, M and N, with the control shown in Supplemental Figure 3), supporting its identification as an AV node. In addition, the sling was observed in one heart in which continuous expression of Tbx3 in both the anterior AV node and the anterior AV bundle (Gja5+) was detected (n = 1/3) (Figure 1, O and P). These results indicate that the left-right axis shapes the disposition of the AV conduction system.

Abnormal cardiac conduction system in the hearts of Cryptic–/– embryos at E18.5. (A–J) Representative images of 3D-reconstructed hearts (A and F) and their original images of in situ hybridization with Hcn4 probes (B–E and G–J) in WT (A–E) (n = 6) and Cryptic–/– (F–J) embryos (n = 4) at E18.5. Upper panels in A and F are ventral views showing the lumen of heart chambers and great vessels, whereas lower panels are right ventral views showing Hcn4 expression in pseudocolors (SA node head, dark green; SA node tail, pale green; venous valves and sinus horn, yellow; AV ring, light blue; AV node and cranioventral nodule, blue; AV bundle and septal branch, red; bundle branches, orange). The levels for each transverse section are shown in the lower panels. (K and L) Right craniodorsal views of the base showing Hcn4 expression in the WT (K) and Cryptic–/– (L) hearts presented in A and F, respectively. (M–P) Expression of Tbx3 (M and O), Myl7 (N), and Gja5 (P) was detected in adjacent transverse sections of a Cryptic–/– heart (different from F), corresponding to the level shown in H. Scale bars: 200 μm. aAVB, (anterior) atrioventricular bundle; aAVN, (anterior) atrioventricular node; Ao, aorta; AVR, atrioventricular ring; cvN, cranioventral nodule; DMP, dorsal mesenchymal protrusion–derived tissue; LA, left atrium; (L/R)BB, (left/right) bundle branch; (L/R)SCV, (left/right) superior caval vein; LSH, left sinus horn; LV, left ventricle; (M)RA, (morphologically) right atrium; PA, pulmonary artery; PV, pulmonary vein; RV, right ventricle; SAN, sinoatrial node; VS, ventricular septum; VV, venous valve.

CCS development in Cryptic–/– embryos. To elucidate the process of abnormal CCS formation in Cryptic–/– embryos, we examined Hcn4 expression at E12.5–E14.5. In the WT atrium at these stages, Hcn4 was continuously expressed in the SA node, venous valves, and dorsal mesenchymal protrusion–derived tissue (42, 43) (Figure 2, A–E, and Supplemental Figure 4, A and B). AV canal–derived tissues, including the AV node, AV rings, and forming AV valves, also expressed Hcn4 (3, 5, 17) (Figure 2, A, D, and E, and Supplemental Figure 4, B and C). Additionally, Hcn4 expression was evident in the caudal IV ring/AV bundle at E12.5 and E14.5 (17) (Figure 2, A, C, and D, and Supplemental Figure 4B). The septal branch derived from the cranial IV ring was faintly and discontinuously detected with the Hcn4 probe at E14.5, but was undetectable at E12.5, whereas an Hcn4-positive cell population was observed in the subaortic portion at E14.5, possibly representing part of the septal branch near the AV ring (Figure 2, A–C, and Supplemental Figure 4A).

Abnormal cardiac conduction system development in the hearts of Cryptic–/– embryos at E12.5–E14.5. (A–J) Representative images of 3D-reconstructed hearts (A and F) and their original images of in situ hybridization with Hcn4 probes (B–E and G–J) in WT (A–E) (n = 8) and Cryptic–/– (F–J) embryos (n = 8) at E14.5, as presented in Figure 1, A–E. The asterisks in F and H show Hcn4 expression in the superior cushion. Scale bars: 200 μm. (K) SA node head volumes of WT and Cryptic–/– embryos at E12.5 (n = 6–7; biological replicates) and E14.5 (n = 4–7; biological replicates). The left and right values of the same embryo are connected. The magenta bars represent mean ± SD. *P < 0.01 (1-way ANOVA followed by Dunnett’s multiple-comparison test). (L) Representative images of voltage mapping with Di-4-ANEPPS in WT (n = 8) and Cryptic–/– hearts (n = 9) at E12.5 (dorsal views). The dotted circles and rectangles indicate the sites where action potential first appeared, around the head and tail of the SA node, respectively. A single heartbeat is shown. (M and N) First breakthrough site of the action potential around the SA node head (M) and atrial side where the action potential first propagated (N). Quantification is based on the final heartbeat from each heart (n = 8–9; biological replicates). Abbreviations are the same as in Figure 1, except for L, left; M, middle; R, right; SB, septal branch.

In Cryptic–/– embryos, the SA nodes were bilaterally developed (n = 8/8 at E14.5; n = 6/6 at E12.5), from which the contiguous expression of Hcn4 extended to the venous valves on the left and right sides (Figure 2, F–J, and Supplemental Figure 4, D and E). Dorsal mesenchymal protrusion formation was markedly impaired (Figure 2I and Supplemental Figure 4E), probably accounting for the development of AVSD (44). Although Hcn4-positive AV rings were detected at E14.5, their cranioventral portions were dorsally bent (Figure 2, F and H). In the superior AV cushion-derived tissue, a distinct Hcn4-positive domain formed a fermata-like mark with the bent AV rings in the transverse sections (n = 7/8) (Figure 2H, asterisk) that continued caudally and tapered into the cushion. The prominent Hcn4-expressing nodule was detected as a protrusion of the cranioventral AV ring at E14.5 (n = 8/8) (Figure 2G). In one heart at E14.5, the cranioventral nodule was apparently continuous with the Hcn4-positive bundle (Supplemental Figure 4F), whereas in others, discontinuous Hcn4 expression in the septal branch was detected (n = 7/8), indicating that a part of Cryptic–/– embryos developed the anterior AV bundle instead of the septal branch.

To evaluate the development and function of the SA node, we first measured the SA node volume using 3D reconstruction of the Hcn4 in situ hybridization sections. The volumes were similar on both sides in Cryptic–/– embryos at E12.5 and E14.5, and were significantly smaller than those in WT embryos (Figure 2K). Cardiac voltage mapping of isolated hearts at E12.5 detected the appearance of action potentials around the SA node, in which simultaneous firing around the head and tail of the SA node (13) was observed in WT (16/36 beats in 8 hearts) and Cryptic–/– (19/37 beats in 9 hearts) hearts (Figure 2L). The first breakthrough site in the craniodorsal portion of the atria was located on the right (n = 7/8) or middle (n = 1/8) of WT hearts, whereas the middle site was significantly more frequent in Cryptic–/– hearts (right: n = 2/9; middle: n = 7/9; P = 0.0152, Fisher’s exact test) (Figure 2M). In addition, while the right atrium propagated action potential earlier than the left atrium in WT hearts (n = 8/8), the first propagated side of the atrium was either the right (n = 4/9) or left (n = 5/9) in Cryptic–/– hearts (P = 0.0294, Fisher’s exact test) (Figure 2N). These changes in the pacemaker position and action potential propagation likely result from bilaterally formed SA nodes, as has been reported for Pitx2 mutant embryos (24, 40). We also recorded successive atrial and ventricular action potential traces in Cryptic–/– hearts at E12.5 by voltage mapping but did not observe any obvious abnormalities compared with WT hearts (Supplemental Figure 5).

Relationship between Pitx2 expression domains and CCS. Because Cryptic is necessary for inducing Pitx2c expression in the left LPM (32, 33), the loss of Pitx2c expression in the heart likely underlies the abnormal CCS in Cryptic–/– embryos. To understand the relationship between CCS-related genes and Pitx2, we compared their expression domains in adjacent sections of WT and Cryptic–/– hearts at E14.5. In WT embryos, Shox2, Tbx3, and Hcn4 colocalized in the SA node and venous valves, which lack Pitx2 expression (Supplemental Figure 6) (3, 12, 19, 20). In Cryptic–/– embryos, Pitx2 expression in the left atrium, left superior caval vein, and pulmonary veins was lost, as expected (Figure 3, A, C, and I–K, and Supplemental Figure 6), whereas Tbx3 and Shox2 were bilaterally expressed in the SA nodes, similar to Hcn4 (Supplemental Figure 6). Notably, Shox2 expression in the pulmonary veins (20) was downregulated, probably because of the absence of a myocardial sleeve, as indicated by the lack of Myl7 expression accompanied by the loss of Pitx2 expression (Supplemental Figure 6) (45).

Pitx2 expression domains in the cardiac conduction system and its cell lineages. (A–N) Transverse sections of hearts from WT (A–H) and Cryptic–/– (I–N) embryos stained by in situ hybridization at E14.5 (A–F and I–M), E12.5 (G and H), and E18.5 (N) for Pitx2 (A, C, E, G–I, K, and M), Tbx3 (B, D, J, L, and N), and Myl7 (F). The arrows indicate Pitx2 or Tbx3 expression in the septal branch (A, B, I, and J) and in the cranioventral side of the AV rings (K). The asterisks in C, D, K, and L indicate the AV bundle, whereas those in E and F denote the AV node. The arrowheads indicate loss of Pitx2 expression in the atrium (I) and left AV ring (M). The dashed rectangle in G indicates Pitx2+ superior AV canal myocardium, including AV rings. The arrows in I and K show residual expression of Pitx2 in the septal branch and AV rings, respectively. (O and P) X-gal staining of a Pitx2 17-Cre CAG-CAT-LacZ heart at E12.0. Coronal sections are shown. The arrow indicates the septal branch. Scale bars: 200 μm. The scale bars in A, G, and O apply to A–F and I–M, G and H, and O and P, respectively. Abbreviations are the same as in Figure 1, except for AS, atrial septum; CAVV, common atrioventricular valve; (i/s)AVC, (inferior/superior) atrioventricular cushion; L, left; MV, mitral valve; R, right; TV, tricuspid valve.

Pitx2 is expressed in the superior/left lateral AV canal and septal branch (25, 27, 29, 46). Concordantly, Pitx2 expression in WT hearts at E14.5 was apparent in the left AV ring and septal branch (Figure 3, A and C), which are the regions where Tbx3 and Hcn4 expression was later downregulated (Figure 2, A–D, and Figure 3, B and D) (8, 12). In contrast, Pitx2 expression was not detected in the AV node and AV bundle (Figure 3, C and E, and Figure 3, D and F, using Tbx3 and Myl7 as CCS markers). At E12.5, the cranioventral portion of the AV rings and cardiomyocytes in the superior AV cushion were Pitx2-positive (Figure 3, G and H), which corresponded to the regions with upregulated Hcn4 expression in Cryptic–/– embryos (Figure 2, F–H). Unexpectedly, in Cryptic–/– embryos, Pitx2 expression was detected in the septal branch at a reduced level (Figure 3, I and J, using Tbx3 as a CCS marker). Pitx2 expression was nearly absent in the left AV ring (Figure 3M), whereas it was sporadically detected in the cranioventral portion of the AV rings (Figure 3K). Consistent with the diminished expression of Pitx2 at E14.5, extensive Tbx3 expression was observed in the anterior AV node and left AV ring of Cryptic–/– embryos (Figure 1M and Figure 3, L and N) (23). Because the superior and left-lateral AV canal is derived from the left second heart field (27, 29, 47), we traced the descendants of Pitx2-expressing cells in the left LPM using the Pitx2 17-Cre transgene, whose enhancer activity depends on Pitx2 ASE regulated by the Nodal signal in the left LPM (29). Embryos from crosses with CAG-CAT-LacZ mice showed X-gal staining in the septal branch and superior AV canal, indicating that both of the regions are derived from the left LPM expressing Pitx2 (Figure 3, O and P).

Pitx2 suppresses CCS formation. The abovementioned results indicated that CCS development is suppressed in regions where Pitx2 is expressed. To confirm this, we analyzed published single-cell RNA-Seq (scRNA-Seq) data from control and Pitx2–/– hearts at E13.5 (48). The uniform manifold approximation and projection (UMAP) analysis identified clusters consistent with those previously published (Supplemental Figure 7, A and B) (48). To enrich putative CCS cells, Tbx3-positive cardiomyocytes were selected and subclustered (Figure 4A and Supplemental Figure 7, A and C). Because Tbx3-positive cells include non-CCS tissues at this stage, CCS clusters were identified with reference to recent scRNA-Seq analyses of CCS at E16.5 (49, 50). Feature and violin plots of the marker genes revealed an SA node cluster (cluster 4, characterized by Cacna2d2+/Shox2+/Smoc2+/Isl1+) (Figure 4, A and B). Notably, the proportions of this cluster were similar between control and Pitx2–/– hearts, suggesting that SA nodes bilaterally formed in Pitx2–/– hearts may be reduced in size, as observed in the Cryptic mutant. Next, we focused on clusters (0, 1, and 5) defined by Cacna2d2+/Kcne1+/Gja1neg (Figure 4, A and B), likely encompassing the compact AV node, nodal AV ring, and AV bundle (49). Subclustering yielded 5 new clusters (A–E) (Figure 4C). The expression profiles of clusters A (Myh6+/Gnao1+/Etv1neg) and E (Myh6hi/Gnao1+/Etv1lo) corresponded to those of the AV ring and compact AV node, respectively (Figure 4D) (49, 50). In Pitx2–/– hearts, the proportion of cluster A decreased (control: 89/9,739 cells vs. Pitx2–/–: 49/8,247 cells), whereas that of cluster E increased (control: 11/9,739 cells vs. Pitx2–/–: 47/8,247 cells), suggesting the presence of dual AV nodes, as observed in Cryptic–/– hearts. Cluster D, characterized by Myh7hi/Irx3+/Etv1+, likely represents the AV bundle/lower nodal cells (Figure 4D) (49). This population was significantly increased in Pitx2–/– hearts (control: 10/9,739 cells vs. Pitx2–/–: 60/8,247 cells), indicative of the development of the anterior AV bundle. Clusters B and C did not correspond to the known CCS components at E16.5 (49), implying that these clusters represent an immature CCS or an abnormally developed population similar to that in the superior AV cushion of Cryptic–/– embryos. Together, these results suggested that AVCS properties were altered in Pitx2–/– embryos.

Altered cardiac conduction system population in Pitx2–/– embryos. (A) UMAP visualization of Tbx3-expressing cardiomyocytes at E13.5 (control, 9739 cells; Pitx2–/–, 8247 cells). The graph shows the proportions of CCS-related cells among the cardiomyocytes: cluster 4 (control, 55 cells; Pitx2–/–, 54 cells); clusters 0, 1, and 5 (control, 155 cells; Pitx2–/–, 327 cells); and the remaining clusters (control, 9,529 cells; Pitx2–/–, 7,866 cells). (B) Expression patterns of Cacna2d2, Smoc2, Shox2, Kcne1, and Gja1 visualized by feature and violin plots derived from the UMAP in A. (C) Subclustering analysis of clusters 0, 1, and 5 from A. The graph shows the ratios of subclusters among the cardiomyocytes: cluster A (control, 89 cells; Pitx2–/–, 49 cells); cluster D (control, 10 cells; Pitx2–/–, 60 cells); and cluster E (control, 11 cells; Pitx2–/–, 49 cells). P values were calculated using the Fisher’s exact test. (D) Feature and violin plots showing the expression of Myh6, Myh7, Kcne1, Gnao1, Irx3, and Etv1 based on the UMAP analysis in C. LNC, lower nodal cells.

Suppressed CCS development in Lefty1–/– embryos. The restricted expression of Pitx2 in the heart suggests that appropriate assignment of Pitx2 expression domains by the left-right axis is fundamental for CCS development. To examine the potential effect of altered Pitx2 expression on CCS formation, we analyzed the CCS of Lefty1–/– embryos using Hcn4 ISH. Lefty1–/– mice develop left isomerism by ectopically expressing Pitx2 to varying degrees in the right LPM (26). Among the 7 Lefty1–/– embryos examined at E14.5, 6 exhibited CHD accompanying left atrial isomerism, including AVSD (n = 5/7), DORV (n = 4/7), TGA (n = 1/7), and intracardiac TAPVC (n = 3/7) (Supplemental Table 1). In Lefty1–/– embryos, the SA node was hypoplastic (E14.5: n = 6/7; E12.5: n = 11/18) (Figure 5, A–D), consistent with the characteristics of left isomerism in humans (34, 36, 37). The volumetry at E12.5 showed that the average SA node volume in Lefty1–/– embryos was significantly reduced in comparison with that in control embryos (Lefty1–/– with CHD: 1.87 ± 0.74 × 10–3 mm3 or Lefty1–/– without CHD: 2.50 ± 0.33 × 10–3 mm3 vs. control: 3.67 ± 0.22 × 10–3 mm3) (Figure 5F). The volumes in Lefty1–/– hearts with CHD varied, likely reflecting the degree of ectopic Pitx2 expression (Figure 5N). Consistent with the hypoplastic SA node, the abnormal propagation of action potential in the atrium was noted in Lefty1–/– embryos at E12.5. Although the difference of first breakthrough site was not significant between control and Lefty1–/– embryos (control: right, n = 11/14; middle, n = 3/14 vs. Lefty1–/–: right, n = 5/9; middle or left, n = 4/9); P = 0.363 from Fisher’s exact test), the ratio that the left-sided atrium first propagated the action potential significantly increased in Lefty1–/– embryos (control: right, n = 14/14 vs. Lefty1–/–: right, n = 6/9; left, n = 3/9; P = 0.0474 from Fisher’s exact test) (Figure 5G and Supplemental Figure 8).

Hypoplastic sinoatrial node and atrioventricular block in Lefty1–/– embryos. (A–E) Representative 3D-reconstructed hearts (A) and corresponding Hcn4 in situ hybridization of a Lefty1–/– embryo (n = 7) (B–E) at E14.5, as in Figure 1, A–E. (F) SA node head volumes in control (Lefty1+/–, Ctl) and Lefty1–/– hearts at E12.5 (n = 4–12; biological replicates). The magenta bars denote mean ± SD. *P < 0.001 (unpaired 2-sided t test with Welch’s correction). (G) Atrial side where the action potential first propagated (n = 9–14). (H) 3D-reconstructed Hcn4 expression in control and Lefty1–/– hearts at E14.5 (different heart from A). The AV conduction systems are viewed from the right craniodorsal side. Asterisks and dotted areas: discontinuous Hcn4 expression at the AV node/AV bundle junction and within the AV bundle, respectively. Arrowhead: continuous expression between the AV bundle and ring. (I) Action potentials in the atrium (Atr) and ventricle (Vnt) of control and Lefty1–/– hearts at E12.5, traced using voltage mapping. (J–M) Transverse sections showing Pitx2 expression in control (J and K) and Lefty1–/– (L and M) hearts. Green bars indicate Pitx2 expression boundary. Arrowheads indicate ectopic Pitx2 expression. Asterisks mark Pitx2 expression in the septal branch. Scale bars: 200 μm; bars in E, J, and K apply to B–D, L, and M, respectively. (N) RT–qPCR of Pitx2c in laser-microdissected heart tissues from control and Lefty1–/– embryos at E12.5, quantified in the SA node region, including the adjacent superior caval vein and right atrial tissue, and in the right caudodorsal AV canal (rcAVC) region. The magenta bars indicate mean ± SD. **P < 0.05 (unpaired 2-sided t test with Welch’s correction). Abbreviations as in Figure 1 except for iAVC, inferior atrioventricular cushion; (L/R)SH, (left/right) sinus horn; MLA, morphologically left atrium.

In control embryos at E14.5 and E12.5, Hcn4 expression in the AV node and bundle/IV ring was continuous (Figure 5H and Supplemental Figure 9). Notably, a discontinuity in Hcn4 expression between the AV node and bundle/IV ring was observed in Lefty1–/– hearts with CHD (n = 2/4 at E14.5; n = 4/9 at E12.5) (Figure 5H and Supplemental Figure 9), which was reminiscent of the dissociation of both components in left isomerism in humans (34, 35). In addition, the staining for Hcn4 was weakened in the AV node of Lefty1–/– embryos at E14.5 (n = 3/4) (Figure 5E). Because discontinuous Hcn4 expression suggests the occurrence of AV block in Lefty1–/– embryos, we analyzed successive action potential in the atrium and ventricle at E12.5 using voltage mapping. Among the 9 Lefty1–/– embryos examined, we detected second-degree (n = 1/9) and third-degree (n = 1/9) AV blocks, whereas such blocks were not observed in control embryos (n = 12/12) (Figure 5I). To correlate the ectopic Pitx2 expression with the CCS of Lefty1–/– embryos, we examined the expression of Pitx2, Hcn4, and Myl7 in adjacent sections at E14.5 (Figure 5, J–M, and Supplemental Figure 10). As expected, Pitx2 was ectopically expressed in the hypoplastic SA node and around the entire AV orifice (n = 2/2) (Figure 5, L and M). In one case, Hcn4 expression in the AV bundle was proximally terminated with a blind end, and its connection to the Myl7+ population, representing the compact AV node, was not observed (Supplemental Figure 10), indicating the disconnection of the AV bundle from the AV node. The ectopic expression of Pitx2c was further confirmed by RT-qPCR of the SA node region and the right caudodorsal region of the AV canal in Lefty1–/– embryos at E12.5 (Figure 5N). Together, these results indicate that the left-right axis determines the disposition and function of the CCS, likely by appropriately defining the expression domains of Pitx2 in the heart.