DEV038562 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Cx30.2-lacZ expression is absent from the developing AV valves. Histological sections taken at high magnification (20×) of the developing AVC region are shown for the indicated time points. Note the β-Gal staining in the AV ring (arrowheads) but not in AV valve progenitor cells of the developing endocardial cushions (arrow).

• Supplemental Figure S2 -

  Fig. S2. Cx30.2 enhancer deletion constructs direct lacZ expression to the AVCS. (A-C) Whole mount (left) and histological sections through the AVN (right, arrow) are shown for the following constructs: −4.0/0.0 (A); −2.9/−1.7 (B); and −2.9/−2.3 (C).

• Supplemental Figure S3 -

  Fig. S3. AVN morphogenesis is unaffected in Gata4^+/− mice. (A) E12.5 embryos with the indicated genotypes were sectioned through the AVC and subjected to radioactive in situ hybridization with a Tbx3 riboprobe. Shown are brightfield (upper panels) and darkfield (lower panels) images for each section. Note that, whereas Tbx3 levels are maintained in Gata4^+/− mice, indicative of preserved AVCS morphology, Tbx3 is diminished in Tbx5^+/− mice (arrow), consistent with its role in normal AVN morphogenesis. (B) E12.5 AVCs were microdissected and subjected to quantitative real-time PCR with a Tbx3 probe, confirming that overall Tbx3 expression is preserved in Gata4^+/− E12.5 AVC tissue. At least three hearts were assayed in triplicate for each genotype. LV, left ventricle; RA, right atrium; RV, right ventricle.

• Supplemental Figure S4 -

  Fig. S4. Gata4^+/− mice have a short PR interval over a wide range of heart rates. EKGs were recorded continuously for 30 seconds on mice stimulated with isoproterenol to elevate the atrial rate. Shown is a scatter plot of heart rate versus PR interval at 10-second intervals for all animals studied with the indicated genotype (wild type, solid circles; Gata4^+/−, open circles). Computer-generated lines were fit to each scatter plot (wild type, solid line; Gata4^+/−, dashed line), demonstrating that the PR interval was shorter in Gata4^+/− mice over a range of different heart rates. The table illustrates the slope (m) and intercept (b) associated with the regression lines along with corresponding P values. ns, not significant.

• Supplemental Figure S5 -

  Fig. S5. Normal PR interval in Gata6^+/− mice. (A) EKG intervals at baseline for Gata6^+/− mice versus their respective wild-type littermates demonstrate that Gata6^+/− mice have normal PR intervals. EKG intervals are represented as mean±s.e.m. (B) EKGs were recorded continuously for 30 seconds on mice stimulated with isoproterenol to elevate the atrial rate as in Fig. 6C. Shown is a scatter plot of heart rate versus PR interval at 10-second intervals for all animals studied with the indicated genotype (wild type, solid circles; Gata6^+/−, open circles). Computer-generated lines were fit to each scatter plot (wild type, solid line; Gata6^+/−, dashed line), showing no significant difference between wild-type and Gata6^+/− mice over a wide range of heart rates. The table illustrates the slope (m) and intercept (b) associated with regression lines along with corresponding P values. ns, not significant.

• Supplemental Figure S6 -

  Fig. S6. Cx40 expression is maintained in the AVC of Gata4^+/− mice. E12.5 AVCs were microdissected and subjected to quantitative real-time PCR with a Cx40 probe, demonstrating that Cx40 expression is unchanged in Gata4^+/− E12.5 AVC tissue compared with wild type. At least three hearts were assayed in triplicate for each genotype. ns, not significant.