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When cardiac function and blood flow are first established are fundamental questions in mammalian embryogenesis. The earliest erythroblasts arise in yolk sac blood islands and subsequently enter the embryo proper to initiate circulation. Embryos staged 0 to 30 somites (S) were examined in utero with 40-to 50-MHz ultrasound biomicroscopy (UBM)-Doppler, to determine onset of embryonic heartbeat and blood flow and to characterize basic physiology of the very early mouse embryonic circulation. A heartbeat was first detected at 5 S, and blood vascular flow at 7 S. Heart rate, peak arterial velocity, and velocity-time integral showed progressive increases that indicated a dramatically increasing cardiac output from even the earliest stages. In situ hybridization revealed an onset of the heartbeat coincident with the appearance of yolk sac-derived erythroblasts in the embryo proper at 5 S. Early maturation of the circulation follows a tightly coordinated program.T he heart is the first organ to develop during embryogenesis. However, little is known about the fundamental questions of when cardiac function and blood flow are first established. Data have come largely from invasive methods 1,2 that disrupt the normal physiological milieu, and scant data exist in the mouse, the primary model of mammalian development. 2,3 Functional circulation is composed of the beating heart, vascular bed, and blood cells. The yolk sac is the earliest and only source of hematopoietic progenitors in the gastrulating mouse embryo. 4 Primitive erythroid progenitors originate in the yolk sac beginning at the late primitive streak stage, before the establishment of circulation in the embryo proper. 4,5 These progenitors give rise to maturing primitive erythroblasts within yolk sac blood islands, which then enter the embryo proper to initiate circulation.We hypothesized that the timing of onset of heartbeat and erythroid cell migration are tightly coordinated. We sought therefore to characterize time points and basic physiology of the very early mouse embryonic circulation using ultrasound biomicroscopy (UBM)-Doppler, a high-frequency echocardiography system developed in our laboratory that allows in utero study of the embryonic mouse circulation. 6 -9 Materials and MethodsThe imaging and whole-mount in situ hybridization protocols have been reported elsewhere, with minor modifications to study earlystage embryos. UBM-Doppler imaging was performed using a semi-invasive in utero approach, exteriorizing the uterus but otherwise maintaining all circulatory systems intact, in close-to-physiological conditions (see the online data supplement available at http://www.circresaha.org for an expanded Materials and Methods section). Results UBM-Doppler Imaging of Early-Stage EmbryosDespite the need for maternal anesthesia, our results indicate that semi-invasive UBM-Doppler yields physiologically relevant data and is uniquely suited to study early embryonic cardiovascular function (see the online data supplement for additional Results). Initiation of Early...

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