Mammalian endothelial cells (ECs) display marked phenotypic heterogeneity. Little is known about the evolutionary mechanisms underlying EC heterogeneity. The last common ancestor of hagfish and gnathostomes was also the last common ancestor of all extant vertebrates, which lived some time more than 500 million years ago. Features of ECs that are shared between hagfish and gnathostomes can be inferred to have already been present in this ancestral vertebrate. The goal of this study was to determine whether the hagfish endothelium displays phenotypic heterogeneity. Electron microscopy of the aorta, dermis, heart, and liver revealed ultrastructural heterogeneity of the endothelium. Immunofluorescent studies demonstrated marked differences in lectin binding between vascular beds. Intravital microscopy of the dermis revealed
IntroductionEndothelial cells (ECs) participate in many physiologic processes, including the regulation of vasomotor tone, hemostasis, leukocyte trafficking, angiogenesis, and permeability. These functions are differentially regulated in space and time (reviewed in Aird 1 ). Although there have been remarkable advances in our understanding of proximate mechanisms of endothelial structure and function in health and disease, far fewer studies have addressed the evolutionary history of this cell lineage.ECs are absent in invertebrates, cephalocordates, and tunicates, but are present in the 3 major groups of extant vertebrates: hagfish (myxinoids), lampreys, and jawed vertebrates (gnathostomes) ( Figure S1, available on the Blood website; see the Supplemental Figure link at the top of the online article). The fact that the endothelium is shared by jawless and jawed vertebrates is evidence that the endothelium was present in the ancestor of these animals. The absence of an endothelium in cephalochordates and tunicates suggests that this structure evolved after the divergence of these groups from the vertebrate lineage, between 540 and 510 million years ago.The last common ancestor of hagfish and gnathostomes was also the last common ancestor of all extant vertebrates, which lived some time more than 500 million years ago. Features of ECs that are shared between hagfish and gnathostomes can be inferred to have already been present in this ancestral vertebrate. Here, we show that hagfish endothelium displays structural, molecular, and functional heterogeneity, suggesting that phenotypic heterogeneity is an ancestral rather than a derived feature of this cell lineage.
Materials and methodsAtlantic hagfish, Myxine glutinosa, were purchased from a commercial supplier (Huntsman Marine Science Center, St Andrews, NB, Canada) who caught the specimens in the Bay of Fundy, Canada. Hagfish were maintained in the dark in circular tanks with running seawater at 10 Ϯ 2°C. The animals were anesthetized in seawater containing tricaine methanesulfonate (MS 222; Sigma-Aldrich, St Louis, MO). For lectin staining, 5-m cryosections were fixed with acetone at Ϫ20°C for 2 minutes and 80% methanol at 4°C for 5 minutes, foll...
