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(Circulation Research. 2008;103:24.)
© 2008 American Heart Association, Inc.

Molecular Medicine

Endothelial Nitric Oxide Synthase Gene Expression During Murine Embryogenesis

Commencement of Expression in the Embryo Occurs With the Establishment of a Unidirectional Circulatory System

Anouk-Martine Teichert, Jeremy A. Scott, G. Brett Robb, Yu-Qing Zhou, Su-Ning Zhu, Melissa Lem, Angela Keightley, Brent M. Steer, Andre C. Schuh, S. Lee Adamson, Myron I. Cybulsky, Philip A. Marsden

From the Renal Division and Department of Medicine (A.-M.T., J.A.S., G.B.R., M.L., A.K., B.M.S., P.A.M.), St. Michael’s Hospital; Department of Medicine (A.-M.T., J.A.S., G.B.R., M.L., A.K., A.C.S., P.A.M.) and Heart & Stroke/Richard Lewar Centre of Excellence in Cardiovascular Research (S.L.A., M.I.C., P.A.M.), University of Toronto; Samuel Lunenfeld Research Institute (Y.-Q.Z., S.L.A.), Mount Sinai Hospital; Mouse Imaging Centre (Y.-Q.Z., S.L.A.), Hospital for Sick Children; Department of Obstetrics and Gynecology (S.L.A.), Samuel Lunenfeld Research Institute, Mount Sinai Hospital; and Toronto General Research Institute (S.-N.Z., M.I.C.), University Health Network, Toronto, Canada.

Correspondence to Philip A. Marsden, MD, Rm 7358, Medical Sciences Building, University of Toronto, 1 Kings College Circle, Toronto, ON, M5S 1A8, Canada. E-mail p.marsden{at}utoronto.ca

To elucidate the role of endothelial NO synthase (eNOS)-derived NO during mammalian embryogenesis, we assessed the expression of the eNOS gene during development. Using transgenic eNOS promoter/reporter mice (with β-galactosidase and green fluorescent protein reporters), in situ cRNA hybridization, and immunohistochemistry to assess transcription, steady-state mRNA levels, and protein expression, respectively, we noted that eNOS expression in the developing cardiovascular system was highly restricted to endothelial cells of medium- and large-sized arteries and the endocardium. The onset of transcription of the native eNOS gene and reporters coincided with the establishment of robust, unidirectional blood flow at embryonic day 9.5, as assessed by Doppler ultrasound biomicroscopy. Interestingly, reporter transgene expression and native eNOS mRNA were also observed in discrete regions of the developing skeletal musculature and the apical ectodermal ridge of developing limbs, suggesting a role for eNOS-derived NO in limb development. In vitro studies of promoter/reporter constructs indicated that similar eNOS promoter regions operate in both embryonic skeletal muscle and vascular endothelial cells. In summary, transcriptional activity of the eNOS gene in the murine circulatory system occurred following the establishment of embryonic blood flow. Thus, the eNOS gene is a late-onset gene in endothelial ontogeny.

Key Words: angiogenesis • embryogenesis • gene transcription • reporter gene • shear stress