The vascular smooth muscle cells of coronary arteries are distinguished from those of the proximal aorta by a number of structural and functional criteria which may include an increased propensity for atherosclerotic transformation. At present, the source of this variation between smooth muscle subpopulations is uncertain. Whilst smooth muscle of the proximal aorta is thought to be derived from neural crest, the origin of coronary vascular smooth muscle remains uncharacterized. We have previously shown that precursors of the coronary vasculature enter the tubular heart on the same day as the epicardial mantle starts to envelop the myocardium and that coronary vessels form by ingrowth of these migratory precursors and not by outgrowth from the aorta (Mikawa and Fischman, 1992). To study the origin of coronary smooth muscle cells, the proepicardial organ, from which epicardial cells arise, was tagged with either a vital dye (DiI) or replication-defective retroviruses encoding beta-galactosidase. Cellular lineage marking was achieved by either direct targeting of putative vasculogenic cells in the proepicardium in ovo or tagging dissected proepicardial cells in vitro followed by transplantation to stage-matched host embryos. Monitoring of tagged cells during coronary vasculogenesis indicate incorporation of proepicardial-derived cells into three vessel-associated populations; coronary smooth muscle, perivascular connective tissue, and endothelial cells. Immunoconfocal microscopy identified both endothelial and smooth muscle cell populations within the proepicardial organ. The results demonstrate that: (1) the proepicardium contains a progenitor population of coronary smooth muscle cells that migrates into the heart along with ingrowth of the epicardium and (2) prior to the migration, the coronary smooth muscle lineage is established.

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SUMMARYCardiac trabeculation is a crucial morphogenetic process by which clusters of ventricular cardiomyocytes extrude and expand into the cardiac jelly to form sheet-like projections. Although it has been suggested that cardiac trabeculae enhance cardiac contractility and intra-ventricular conduction, their exact function in heart development has not been directly addressed. We found that in zebrafish erbb2 mutants, which we show completely lack cardiac trabeculae, cardiac function is significantly compromised, with mutant hearts exhibiting decreased fractional shortening and an immature conduction pattern. To begin to elucidate the cellular mechanisms of ErbB2 function in cardiac trabeculation, we analyzed erbb2 mutant hearts more closely and found that loss of ErbB2 activity resulted in a complete absence of cardiomyocyte proliferation during trabeculation stages. In addition, based on data obtained from proliferation, lineage tracing and transplantation studies, we propose that cardiac trabeculation is initiated by directional cardiomyocyte migration rather than oriented cell division, and that ErbB2 cellautonomously regulates this process.

A new simple method of detecting calcium binding proteins in a protein mixture is described. A sample which might include calcium binding proteins was subjected to SDS-polyacrylamide gel electrophoresis and then electrophoretically transferred to a nitrocellulose membrane. The membrane was then incubated with 45Ca to detect calcium binding proteins as radioactive bands by autoradiography. Purified troponin-C, calmodulin, myosin DTNB light chain, and parvalbumin were clearly identified by this method. In the whole homogenate of chicken skeletal muscle, myosin DTNB light chain, troponin-C, and 55K calcium binding protein were found to be radioactive. In the frog skeletal muscle, small molecular weight proteins of approximately 13-15K and 70K protein appeared to be the calcium binding proteins. In the case of the carp skeletal muscle, small molecular weight proteins including parvalbumin and two proteins of about 80K seemed to bind calcium ion. Two high molecular weight calcium binding proteins were present in the scallop striated muscle. The procedure described can be completed within 24 h and can detect as little as 2 micrograms of calcium binding protein in the starting sample. Under appropriate conditions it was possible to detect only high affinity calcium binding proteins.

We associated environmental and genetic factors with base substitution patterns of somatic mutations and provide a registry of genes and pathways that are disrupted in ESCCs. These findings might be used to design specific treatments for patients with esophageal squamous cancers.