Onset of Cardiac Function During Early Mouse Embryogenesis Coincides With Entry of Primitive Erythroblasts Into the Embryo Proper
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.
The 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 methods1,2 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 Methods
The imaging and whole-mount in situ hybridization protocols have been reported elsewhere, with minor modifications to study early-stage 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).
UBM-Doppler Imaging of Early-Stage Embryos
Despite 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 Cardiovascular Function and Correlation With Erythroblast Entry
The onset of heartbeat and blood flow were precisely orchestrated over a ≈5-hour window from 5 S to 8 S (Figure 1). At the earliest periods of cardiac activity, the heart could be seen by UBM to contract rhythmically, without detectable embryonic blood flow elsewhere. Even the earliest heartbeats appeared regular. Later, “speckle” blood flow in the heart and along the long axis of the embryo (in the presumed aorta), as well as cardiac motion, could be discerned. Aortic Doppler flow began by 7 S and was consistently obtained by 8 S.
Before initiation of the heartbeat (2 S to 4 S), no intraembryonic erythroblasts were detected by in situ hybridization (Figure 2). With the onset of cardiac contractions, scattered erythroblasts were observed within the head region and aorta of the embryo. Progressively more erythroblasts were evident in embryos exhibiting speckle and Doppler flow. Thus, initial movement of erythroblasts into the embryo proper coincided with the onset of heartbeat at 5 S to 6 S, and maturation of cardiovascular function and erythroblast density occurred together.
Because early, primitive hematopoiesis is confined to the yolk sac blood islands before ≈E11 to E12,4,5 the presence of blood cellular flow within the embryo implied a connection between intra- and extraembryonic circuits. We therefore corroborated our results by examining the timing of vitelline (yolk sac) blood flow. Embryos displaying no heartbeat or heartbeat only failed to show any vitelline flow along the yolk sac. Of 8 embryos displaying intraembryonic speckle flow without Doppler, vitelline flow was seen in 3, observed as early as 5 S. Finally, 14 of 15 embryos with Doppler signals exhibited vitelline flow, seen at ≥7 S to 8 S. Vitelline flow thus correlated with onset of other cardiac functional parameters.
Early Cardiovascular Physiology
Heart rates at 5 S to 6 S were estimated using UBM to be 100 to 130 bpm. At later stages, Doppler-derived heart rates increased from 137±19 bpm at 7 S to 8 S, to 173±16 bpm at 21 S to 30 S (P<0.002) (Figure 3A). Peak arterial velocity and arterial velocity-time integral, which reflect cardiac stroke volume,8,9 also increased markedly (P<0.03) (Figure 3B). Even early-stage Doppler waveforms closely resembled those of the more mature organism (Figure 2).
Doppler-derived time intervals provided further insight into early cardiovascular function (Figure 3C). Acceleration time, an index of myocardial contractility influenced by heart rate,8 decreased significantly through E10.5 (P<0.01). Although ejection time did not change (P>0.60), nonejection time and cycle length decreased in parallel (P<0.003). Therefore, ejection time as a proportion of cycle length, a measure of systolic cardiac work, increased (P<0.05, data not shown). Our collective results argue for a dramatic increase in cardiac stroke work and output from even the earliest stages of cardiac development.
The present study is the first to demonstrate that the onset of in vivo rhythmic cardiac contractions coincides with the initial appearance of erythroblasts in the mammalian embryo proper. Our study also provides the first quantitative insights into cardiovascular mechanics in the nascent circulation, during a period of rapid cardiac formation.
Initiation of the Heartbeat
Only scant previous data are available on the initiation of cardiac contraction in the mammalian embryo, particularly the mouse.1,2,7,10,11 In the mouse, a patent endocardial tube is formed at ≈6 S, the primitive heart is linked to the intact dorsal aortas by 5 S to 7 S, and the heart appears “capable of maintaining some circulation of the blood” (page 44)10 at 13 S to 20 S.10,12 Other investigators have noted weak, irregular cardiac contractions at 3 S to 4 S, with “fairly regular” (page 78)2 contractions by 5 S to 6 S, but circulation was not assessed.2
Although undoubtedly we are not observing the very first contractions of cardiomyocytes, which likely involve only a few cells, we are observing very early whole-organ contraction with UBM. Cardiac contractions commence even before cardiac looping.1,2,10,12 The timing of the initial movement of erythroblasts from yolk sac to embryo proper, coincident with the onset of cardiac contractions, is consistent with recent work.13 The requirement of a heartbeat first to initiate and subsequently to maintain erythrocyte circulation has been strongly suggested in mouse models lacking a heartbeat.14 It thus appears that yolk sac blood island and intraembryonic vascular networks are connected when cardiac contractions commence, and that erythroblasts cannot migrate actively into the embryo proper before commencement of the heartbeat with presumably acellular blood flow. Our results thus establish the early timing of the initiation of the heartbeat and circulation in the mouse.
Physiology of the Very Early Circulation
Parameters of cardiac output and stroke work here align neatly with later-stage data previously reported from our laboratory.8,9 Our results that early Doppler waveforms closely resemble those in the more mature organism8,15 indicate a relative strength and velocity of contraction that belie the seemingly disorganized myofibrils in these early hearts.2 The nascent heart thus seems capable of dramatic increases in stroke work and output, even when it is little more than a tube.
An interesting question is raised by the suggestion of a markedly increasing cardiac output, at stages when a functional circulation is not required for embryonic viability. For example, mice lacking a heartbeat, a properly developed vascular bed, or erythroid cells die at ≈E10 to E11.14,16,17 Our results indicate that early development of the circulation follows a tightly coordinated program that brings together maturing red cells, an intact vascular network, and cardiac function over a 48-hour period (≈E8 to E10) to provide for continued growth of the mammalian embryo.
This work was supported by an American Heart Association, Heritage Affiliate, Scientist Development Grant 0030333T (C.K.L.P.) and NIH grants HL04414 (C.K.L.P.), HL59484 (J.P.), and NS38461 (D.H.T.). We thank Jeff Malik for his excellent technical assistance.
↵*Both authors contributed equally to this study.
Original received October 4, 2002; resubmission received December 6, 2002; revised resubmission received January 6, 2003; accepted January 6, 2003.
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