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.

Isl1(+) cardiovascular progenitors and their downstream progeny play a pivotal role in cardiogenesis and lineage diversification of the heart. The mechanisms that control their renewal and differentiation are largely unknown. Herein, we show that the Wnt/beta-catenin pathway is a major component by which cardiac mesenchymal cells modulate the prespecification, renewal, and differentiation of isl1(+) cardiovascular progenitors. This microenvironment can be reconstituted by a Wnt3a-secreting feeder layer with ES cell-derived, embryonic, and postnatal isl1(+) cardiovascular progenitors. In vivo activation of beta-catenin signaling in isl1(+) progenitors of the secondary heart field leads to their massive accumulation, inhibition of differentiation, and outflow tract (OFT) morphogenic defects. In addition, the mitosis rate in OFT myocytes is significantly reduced following beta-catenin deletion in isl1(+) precursors. Agents that manipulate Wnt signals can markedly expand isl1(+) progenitors from human neonatal hearts, a key advance toward the cloning of human isl1(+) heart progenitors.

A number of human congenital disorders present with both heart and limb defects, consistent with common genetic pathways. We have recently shown that the LIM homeodomain transcription factor islet 1 (Isl1) marks a subset of cardiac progenitors. Here, we perform lineage studies with an Isl1Cre mouse line to demonstrate that Isl1 also marks a subset of limb progenitors. In both cardiac and limb progenitors, Isl1 expression is downregulated as progenitors migrate in to form either heart or limb. To investigate common heart-limb pathways in Isl1-expressing progenitors, we ablated the Type I Bmp receptor, Bmpr1a utilizing Isl1Cre/+. Analysis of consequent heart and limb phenotypes has revealed novel requirements for Bmp signaling. Additionally, we find that Bmp signaling in Isl1-expressing progenitors is required for expression of T-box transcription factors Tbx2 and Tbx3 in heart and limb. Tbx3 is required for heart and limb formation, and is mutated in ulnar-mammary syndrome. We provide evidence that the Tbx3 promoter is directly regulated by Bmp Smads in vivo.