Islet 1 is expressed in distinct cardiovascular lineages, including pacemaker and coronary vascular cells

Abstract

Islet1 (Isl1) is a LIM homedomain protein that plays a pivotal role in cardiac progenitors of the second heart field. Here, lineage studies with an inducible isl1-cre demonstrated that most Isl1 progenitors have migrated into the heart by E9. Although Isl1 expression is downregulated in most cardiac progenitors as they differentiate, analysis of an isl1-nlacZ mouse and coimmunostaining for Isl1 and lineage markers demonstrated that Isl1 is expressed in distinct subdomains of the heart, and in diverse cardiovascular lineages. Isl1 expression was observed in myocardial lineages of the distal outflow tract, atrial septum, and in sinoatrial and atrioventricular node. The myocardialized septum of the outflow tract was found to derive from Isl1 expressing cells. Isl1 expressing cells also contribute to endothelial and vascular smooth muscle lineages including smooth muscle of the coronary vessels. Our data indicate that Isl1 is a specific marker for a subset of pacemaker cells at developmental stages examined, and suggest genetic heterogeneity within the central conduction system and coronary smooth muscle. Our studies suggest a role for Isl1 in these distinct domains of expression within the heart.

Fig. 1

Lineage analysis of embryos doubly heterozygous for isl1-MerCreMer and R26RlacZ by tamoxifen induction. (A) Generation of Isl1 MerCreMer knock-in mouse (a) Targeting strategy. The relevant genomic region of isl1 is shown on top, the targeting construct is shown in the center, and the locus after recombination is shown at the bottom. A knock-in cassette composed of MerCreMer followed by FRT-mclNeo was introduced at SalI site in isl1 5'-untranslated region. Red box represents the MerCerMer cDNA and gray box represents the mcl-neomycin gene. (b) Detection of wild-type and targeted alleles by Southern blot analysis. DNAs from neomycin-resistant ES clones were digested with BamHI and analyzed by Southern blot with probe as shown in A-a. The 10.5- and 7.5-kb bands represent wild-type and targeted alleles, respectively. (B). Embryos injected at E6 and harvested at E8 revealed that labeled cells had already migrated into the early linear heart tube at both anterior and posterior poles, contributing to both myocardium and endocardium. (C) Corresponding sections of ED8.5 (8 somites) embryos are shown, progressing from anterior to posterior and (D) Corresponding sections of ED8.5 (10 somites) embryos are shown, progressing from anterior to posterior. Arrows indicate positive cells in endocardium. (E, F) Embryos injected at E7 and harvested at E9 and (G, H) Embryos injected at E8 and harvested at E10 showed labeled cells in outflow tract, atria, ventricle, and within mesenchymal cells of the atrioventricular (AV) canal (positive cells indicated by arrows and negative cells indicated by arrowheads). (I, J) Inductions performed at E9 revealed positive staining in the most distal cells of the outflow tract and a distinct subset of cells in the atria, in the region of the sinoatrial node and atrioventricular node (indicated by arrows). A: atria; AVN: atrioventricular node; LA: left atria; LV: left ventricle; OT: outflow tract; RA: right atria; LV: left ventricle; SAN: sinoatrial node. The framed areas were shown by high magnification in next photos.

Fig. 2

Analysis of β-galactosidase expression in isl1-nlacZ knock-in embryos using X-gal staining. (A) Generation of isl1-nlacZ knock-in mice. (a) Targeting strategy is the same as that of isl1-MerCreMer, except that the knock-in cassette is a Sall fragment of Lox-P-nLacZ followed by hrGFP and FRT-mclNeo gene. Colored boxes represent: blue box, nLacZ, which is flanked by Lox-P, green box, hrGFP and gray box,mcl-neomycin resistance gene, which is flanked by FRT. (b) Detection of wild-type and targeted alleles by Southern blot analysis as described in A-a. The 10.5- and 7.5-kb bands represent wild-type and targeted alleles, respectively. (B) Embryos at E8.5 stained with X-gal in left, frontal, and right views revealed active expression of β-gal in foregut endoderm, splanchnic mesoderm. (C) Corresponding sections of ED8.5 (8 somites) embryos are shown, progressing from anterior to posterior, (D) Corresponding sections of ED8.5 (10 somites) embryos are shown, progressing from anterior to posterior, (E) Embryos at E9.5 stained with X-gal in left and right views, and (F) Corresponding sections from anterior to posterior revealed isl1-nlacZ expression within outflow tract, right atria and right ventricle. Arrows indicate positive cells in endocardium. (G) Embryos at E11.5 stained with X-gal in left and right views, and (H) Corresponding sections from anterior to posterior revealed isl1-nlacZ expression within outflow tract, atrial septum, and sinoatrial and atrioventricular nodes. (I) Heart from E14.5 embryos stained with X-gal in ventral, dorsal and lateral dorsal views, and (J) Corresponding sections from ventral to dorsal revealed isl1-nlacZ expression within outflow tract, aorta, pulmonary artery, atrial septum, venous valves, cardiac ganglia and sinoatrial and atrioventricular nodes. (K) At postnatal day 3, Isl1-nlacZ expression was observed in cardiac ganglia, the region of the sinoatrial node and at the base of the aorta/pulmonary artery. A: atria; AS: atria septum; AVN: atrioventricular node; CG: cardiac ganglia, LA: left atria; LV: left ventricle; OT: outflow tract; RA: right atria; SAN: sinoatrial node.

Fig. 3

Expression of isl1-nlacZ mirrors endogenous Isl1 expression. (A-H) Coimmunostaining with antibodies to Isl1 and β-galactosidase (β-gal) on cardiac sections of isl1-nLacZ knock-in embryos at E11.5. (I-K) Co-immunostaining of Isl1 and β–gal antibodies on cardiac sections of isl1-nLacZ knock-in embryos at E13.5. (L-T) Co-immunostaining of Isl1 and β-gal antibodies on postnatal day 3 cardiac sections of isl1-lacZ knock-in mouse.

Fig. 4

Isl1 expression in myocardial lineages and cardiac conduction system. (A-D) Coimmunostaining of isl1 or β-gal with a monoclonal antibody to muscle specific myosin heavy chain, MF20, on sections from E11.5 (A-D) and E14.5 (I-L) embryos revealed expression of isl1 in outflow tract myocardium. Co-immunostaining of isl1 or β-gal and α-smooth muscle actin on sections from E11.5 (E-H) and E14.5 (M-P) embryos revealed expression of isl1 in outflow tract myocardium. (Q-T) Co-immunostaining for isl1 on sections obtained from an HCN4-GFP knock-in mouse revealed expression of isl1 in subsets of HCN4+ populations in the regions of SA node, AV node and atrial septum (arrow). Co-immunostaining of isl1 on E13.5 sections demonstrated that Isl1 expressing cells in atrial septum co-expressed Nkx2.5 (U-X), and α-actinin (Y-B′), the latter a marker for differentiated myocytes.

Fig. 5

Isl1 expression in endothelial lineages. Co-immunostaining for Isl1 and PECAM-1 showed expression of Isl1 protein in PECAM positive endothelial cells within outflow tract at E11.5 (A-E) and in endothelial cells within aorta and pulmonary artery at E12.5 (F-I).

Fig. 6

Isl1 expression in smooth muscle lineages. (A-D) Coimmunostaining for Isl1 and α-smooth muscle actin in sections from E12.5 embryos showed colocalization in proximal aorta and pulmonary artery trunk. (E-L) In the heart from postnatal day 0 (PN0) isl1-cre;R26RlacZ mouse, β-gal expressing cells co-expressed α-smooth muscle actin in coronary vasculature in Left (E-H) and right (I-L) ventricles but not in eipcardium (arrow in H and L) .

Fig. 7

Isl1 expression in cardiac neural crest lineages. (A-D) Co-immunostaining for Isl1 and β-gal on tissue sections from Wnt1-cre;R26RlacZ embryos at E12.5 showed that only a small number of Isl1- and β–gal-positive cells overlapped in the outflow tract (arrows indicate overlapping staining). (E-H) Co-immunostaining for Isl1 and neurofilament on tissue sections from E13.5 embryos revealed Isl1 expression in cardiac ganglia.