Enhanced Proliferation of Monolayer Cultures of Embryonic Stem (ES) Cell-Derived Cardiomyocytes Following Acute Loss of Retinoblastoma

Background: Cardiomyocyte (CM) cell cycle analysis has been impeded because of a reliance on primary neonatal cultures of poorly proliferating cells or chronic transgenic animal models with innate compensatory mechanisms. Methodology/Principal Findings: We describe an in vitro model consisting of monolayer cultures of highly proliferative embryonic stem (ES) cell-derived CM. Following induction with ascorbate and selection with puromycin, early CM cultures are.98 % pure, and at least 85 % of the cells actively proliferate. During the proliferative stage, cells express high levels of E2F3a, B-Myb and phosphorylated forms of retinoblastoma (Rb), but with continued cultivation, cells stop dividing and mature functionally. This developmental transition is characterized by a switch from slow skeletal to cardiac TnI, an increase in binucleation, cardiac calsequestrin and hypophosphorylated Rb, a decrease in E2F3, B-Myb and atrial natriuretic factor, and the establishment of a more negative resting membrane potential. Although previous publications suggested that Rb was not necessary for cell cycle control in heart, we find following acute knockdown of Rb that this factor actively regulates progression through the G1 checkpoint and that its loss promotes proliferation at the expense of CM maturation. Conclusions/Significance: We have established a unique model system for studying cardiac cell cycle progression, and show in contrast to previous reports that Rb actively regulates both cell cycle progression through the G1 checkpoint an

Data from: Demographic history influences spatial patterns of genetic diversity in recently expanded coyote (Canis latrans) populations

Human-mediated range expansions have increased in recent decades and represent unique opportunities to evaluate genetic outcomes of establishing peripheral populations across broad expansion fronts. Over the past century, coyotes (Canis latrans) have undergone a pervasive range expansion and now inhabit every state in the continental United States. Coyote expansion into eastern North America was facilitated by anthropogenic landscape changes and followed two broad expansion fronts. The northern expansion extended through the Great Lakes region and southern Canada, where hybridization with remnant wolf populations was common. The southern and more recent expansion front occurred approximately 40 years later and across territory where gray wolves have been historically absent and remnant red wolves were extirpated in the 1970s. We conducted a genetic survey at 10 microsatellite loci of 482 coyotes originating from 11 eastern U.S. states to address how divergent demographic histories influence geographic patterns of genetic diversity. We found that population structure corresponded to a north-south divide, which is consistent with the two known expansion routes. Additionally, we observed extremely high genetic diversity, which is atypical of recently expanded populations and is likely the result of multiple complex demographic processes, in addition to hybridization with other Canis species. Finally, we considered the transition of allele frequencies across geographic space and suggest the mid-Atlantic states of North Carolina and Virginia as an emerging contact zone between these two distinct coyote expansion fronts