Fig. 5

Figure 5. Deletion of the Isl1 sinoatrial node (SAN) enhancer leads to reduced Isl1 expression in SAN and abnormal SAN development.A, Top, Deletion of a 2.7-Kb genomic segment containing the Isl1 locus sinoatrial node enhancer (ISE). Middle, Genotyping strategy to identify the mutant allele with an agarose gel (bottom) on an F4 intercross confirming germline transmission of the deletion allele. F denotes forward primer; and REV denotes reverse primer. B, Genotypes from 88 weaned pups of Isl1^?ISE/+ intercrosses with the indicated frequencies. C, Immunohistochemistry of wild-type (WT; top) and Isl1^?ISE/?ISE (bottom) SAN at embryonic day (E) 14.5 stained for Hcn4 (hyperpolarization-activated cyclic nucleotide-gated ion channel 4; red), Isl1 (Islet-1; green), and DAPI (4',6-diamidino-2-phenylindole). Area of detail is shown in the right parts. Pacemaker cell nuclei in the WT are Isl1+ (turquoise, denoted by white arrows) whereas the Isl1^?ISE/?ISE has less Isl1 signal in pacemaker cell (PC) nuclei. D, Quantification of green fluorescent signal (Isl1) in 35 Hcn4+ pacemaker cell nuclei and 35 Hcn4- non-PC nuclei, normalized to tissue background green fluorescent signal, from 7 sections each from 2 different sets of WT and Isl1^?ISE/?ISE littermates (denoted replicate 1 and replicate 2). ****P<0.0001 and ***P<0.001. E, Junction of the superior vena cava (svc) and right atrium (ra) showing the location of the sinoatrial node (san) and the sectioning plane used to perform PC counts and cross-sectional area measurements. Z=0 denotes the center of the sinoatrial node head. F, Quantification of PC nuclei in 12-sections each at the indicated z-distance from the center of the SAN head. Counts were performed for 2 pairs of E14.5 littermates (denoted replicate 1 and replicate 2). G, Estimation of area of the SAN head for the sections analyzed in F. *P<0.05. NS indicates nonsignificant