(A) X-gal staining of an E11.5 Dct-LacZ embryo demonstrated staining near pharyngeal arches (arrows), developing telencephalon (T), and eye (E). (B) In situ hybridization of an E13.5 heart showed Dct expression (filled arrows) around a PV os (open arrowhead). (C) X-gal staining of an E13.5 Dct-LacZ heart in the posterior atrium and endocardial cushions. (D) X-gal staining of an E13.5 Dct-LacZ heart in the septal leaflet valve primordia. Inset scale bar: 500 μm. (E) Immunofluorescence of Dct-positive cells (arrowheads) within the PVs. (F) X-gal staining in an E16.5 Dct-LacZ heart in the posterior atrium (arrows). (G and H) X-gal staining in a Dct-LacZ heart along the right atrial septum above the valve annulus at E17.5 (G; inset scale bar: 500 μm) and P1 (H). (I) Immunofluorescence showed Dct-positive cells lining the SAN artery epithelium, the tricuspid valve, and the mitral valve. Inset (scale bar: 100 μm) shows boxed region. (J) Immunofluorescence of Dct-positive cells (arrow) in the tricuspid valve of a 15-day-old mouse. (K) Adult mouse (P60) resulting from crossing a DctCre^+/– and R26R mouse demonstrated LacZ-positive cells (arrows) within the PVs and atria. (L) Dct-positive cells detected by immunofluorescence (arrows) on the endothelium of explanted human PV. Scale bars: 500 μm (A–C, F, H, I, K); 100 μm (D, E, G); and 50 μm (J and L). LA, left atrium; Ao, aorta; PA, pulmonary artery; MV, mitral valve; TV, tricuspid valve; RA, right atrium; IVC, inferior vena cava; FO, foramen ovale; CS, coronary sinus; SAN, sinoatrial node; VV, venous valve.

(A–O) Sections from E17.5 hearts co-stained by immunofluorescence for Dct (A, D, G, J, and M), Sox10 (B), Mitf (E), Tyr (H), Pax3 (K), and Tyrp-1 (N), respectively. Merged images for Dct and each individual marker are shown (C, F, I, L, and O). (P–R) Section through the anterior leaflet of the pulmonary valve of an E17.5 Wnt1Cre, R26R heart stained for Dct (arrows, P) and β-galactosidase (open arrows, Q). No overlap of staining was seen in merge (R). Original magnification, ×400 (A–O); ×100 (P–R).

(A) Low-power and (B) high-power transmission electron microscopy demonstrated association between 4 Dct-LacZ–expressing cells. White arrowheads indicate electron-dense X-gal precipitate in the cytoplasm and perinuclear area. N denotes the same nucleus in both A and B (scale bars: 2 μm and 1 μm, respectively). (C and D) High-power transmission electron microscopy of 2 Dct-expressing cells. Cells in C contained no melanosomes, while Cell2 in D contained type III and type IV melanosomes. Cell1 contained X-gal precipitate (white arrowhead). Black arrowheads in C and D denote caveolae. Scale bars: 500 nm. (E and F) High-power transmission electron microscopy of mitochondria in the atrial myocardium of a Dct^+/– mouse (E) and a Dct^–/– mouse (F). Note that some mitochondria appear slightly enlarged, with less densely packed and disorganized matrices in the absence of Dct. Scale bars: 500 nm. N, nucleus; M, mitochondria; III, type 3 melanosome; IV, type 4 melanosome.

Each column represents expression data from a single cell from 1 of 3 classes: cardiac Dct-expressing cell (pink), atrial myocyte (orange), or dermal Dct-expressing cell (green). Expression analysis was performed on those transcripts that had 2-fold expression above or below the mean in each cell class. The cluster analysis depicted at top was subsequently performed after ANOVA demonstrated significant expression differences between each of the 3 cell classes. Fold changes are represented graphically, with red tiles demonstrating greater than 2-fold expression above the median, yellow representing median expression, and blue demonstrating expression 2-fold below the median. See Supplemental Table 3 for the identity of the genes analyzed. Dendrograms show correlation of gene expression profiles between samples. The length of the dendrogram branches connecting pairs of nodes represents the level of correlation between samples. Note that each cell class segregates to a discrete node, with the cardiac Dct-expressing cells sharing greater correlation with atrial myocytes than dermal Dct-expressing cells, confirming that each cellular class has a distinct signature.

Hearts derived from Dct^+/– (A and C) or Dct^–/– (B and D) mice that also carried the Dct-LacZ transgene were stained with X-gal. Anterior (A and B) and posterior (C and D) views are shown, with labeled cells seen in a similar pattern in Dct^+/– and Dct^–/– (filled arrows). Insets in C and D show the posterior atrial wall, where labeled cells are evident (arrows). Original magnification, ×40 (A–D); ×100 (insets).

(A) Recordings from ECG lead I (I), right atrium, and right ventricle show AF induced by burst stimulation (S) in a Dct^–/– animal. Irregularly irregular intervals between ventricular (v) beats are denoted. Sinus beats are shown on termination of the episode with normal atrial (a) and ventricular electrograms. Inset demonstrates magnified view of denoted area. (B) Real-time telemetry recorded in an awake Dct^–/– mouse with an implantable monitor demonstrates spontaneous AF characterized by abrupt onset of a rapid irregularly irregular rhythm with no discrete p-waves. The inset shows a faster time scale of a few beats of sinus rhythm (arrows), followed by the onset of AF. (C) Surface ECGs from Dct^–/– and Dct^+/– littermate mice demonstrating normal ECG morphologies. (D) Two-dimensional echocardiographic imaging from a 4-chamber view demonstrating no qualitative difference in atrial dimensions during atrial systole or atrial diastole in Dct^–/– and Dct^+/+ littermate mice. Scale bar: 2 mm.

Shown are representative hearts from wild-type (A–D), Wv/+ (E–H), W/+ (I–L), and W/Wv (M–P) littermates. The wild-type mouse demonstrated normal dermal pigmentation (A and B) and the presence of cardiac pigmentation (C and D). Tricuspid valve at low (C) and high power (D); black arrow denotes pigmented cells in a valve leaflet. Wv/+, W/+, and W/Wv mice showed abnormal dermal pigmentation (E, F, I, J, M, and N) as well as absence of pigmented cells within the valves (G, H, K, L, O, and P). Immunohistochemical staining with an anti-Dct antibody similarly showed labeling within wild-type mouse hearts, but cardiac staining was absent in Wv/+, W/+, and W/Wv mice (not shown). Original magnification, ×40 (C, G, K, and O); ×200 (D, H, L, and P).

Immuno­histochemistry within the adult mouse atrium using an antibody to Dct demonstrated Dct-positive cells with characteristic morphology (A, D, G, J, and M). Sections were co-stained with antibodies to β1-adrenergic receptor (β1R; B), α1-adrenergic receptor (α1R; E), muscarinic receptor subtype 3 (M3R; H), tyrosine hydroxylase (T-OH; K), and choline acetyltransferase (ChAT; N). Merged images of Dct and each respective antibody are shown, demonstrating coexpression of Dct with β1R (C), α1R (F), and M3R (I) receptors (arrowheads). Additionally, presumptive nerve terminals that express tyrosine hydroxylase (L) and choline acetyltransferase (O) were seen in close proximity to Dct-expressing cells (arrows). Scale bars: 20 μm.

(A) Current clamp recordings from cardiac Dct^+/– and Dct^–/– cells demonstrated increased action potential duration in the absence of Dct. Resting potential of the Dct^+/– cell was –62 mV, while the Dct^–/– cell had a resting potential of –60 mV. (B) Representative cardiac melanocyte from a Dct^+/–, Z/EG mouse visualized by DIC and fluorescence (GFP). Scale bar: 10 μm. (C) Spontaneous action potential recordings from Dct^–/– and Dct^+/– cardiac melanocytes. Within the trace from the Dct^–/– cell, note the presence of an early afterdepolarization (arrow) arising from phase 3 of the preceding action potential. The trace from a Dct^+/– cell is included for comparison.

(A–D) Representative cells from Dct^+/–, Z/EG and Dct^–/–, Z/EG hearts. A and C show DIC images; B and D show DIC images merged with fluorescent images demonstrating GFP-positive Dct-expressing cells. Mel, Dct-expressing cell; myo, atrial myocyte. Scale bars: 20 μm. (E and F) Representative linescans normalized to background from Dct^+/– (E) and Dct^–/– (F) preparations demonstrated calcium mobilization in the Dct-expressing cell (mel) as well as an adjacent myocyte (myo). (G–K) Graphs demonstrating normalized calcium flux in a Dct^+/– melanocyte (G) and an adjacent myocyte (J), as well as from a Dct^–/– melanocyte (H) and an adjacent myocyte (K). Note an oscillation following the fourth complex in H characterized by a rapid upstroke prior to full repolarization of the preceding complex. (I) Portions of the graph in H and K, denoted by the horizontal red bar, at a higher time resolution. Note that the upstroke of the oscillation in the melanocyte precedes calcium mobilization in the adjacent myocyte (vertical red bar). F/F₀, measured fluorescence normalized to peak fluorescence.

(A–C) A lucifer dye transfer experiment demonstrated functional coupling between a Dct-expressing cell and an adjacent atrial myocyte in culture. (A) A patch pipette containing 250 μM of lucifer yellow in the cell-attached configuration prior to membrane rupture (time 1). (B and C) Dct-expressing cardiac melanocyte in the whole-cell configuration after membrane rupture (time 2) visualized with fluorescence excited at 488 nm (B) and with both fluorescence and white light via Hoffman illumination (C). (D–F) Dct-positive cells in the atrium express connexin 45 (Cx45). Sections from an adult wild-type mouse atrium co-stained by immunofluorescence with Dct (D) and connexin 45 (E). Dct-positive cells that coexpressed connexin 45 in the merge image (F) are indicated by arrowheads. Scale bar: 20 μm.