Ultrasound biomicroscopy-Doppler in mouse cardiovascular ...€¦ · review Ultrasound...

review

Ultrasound biomicroscopy-Doppler in mousecardiovascular development1

Colin K. L. Phoon1,2 and Daniel H. Turnbull1,3

1Skirball Institute of Biomolecular Medicine, 2Pediatric Cardiology Program,Department of Pediatrics, and 3Departments of Radiology and Pathology,New York University School of Medicine, New York, New York 10016

Submitted 16 January 2003; accepted in final form 4 April 2003

Phoon, Colin K. L., and Daniel H. Turnbull. Ultrasound biomi-croscopy-Doppler in mouse cardiovascular development. Physiol Genom-ics 14: 3–15, 2003; 10.1152/physiolgenomics.00008.2003.—The ability tomodify the mouse genome has yielded new insights into the geneticcontrol of mammalian cardiovascular development. However, it is farless understood how genetic factors and their consequent structuralchanges alter cardiovascular function, a void largely due to the lack ofeffective noninvasive techniques to assess function in the developingmouse cardiovascular system. In this review, we discuss the recentadvances in ultrasound biomicroscopy (UBM)-Doppler echocardiographyfor analyzing cardiovascular function in the embryonic mouse in utero.“Cardiovascular function” encompasses broad aspects of physiology, in-cluding systolic and diastolic cardiac function, distribution of blood flowamong various embryonic vascular beds, and vascular bed properties(impedance). A wide range of physiological measurements is possibleusing UBM-Doppler, but it is clear that the limitations of any singlemeasurement warrant a multi-parameter approach to characterizingcardiovascular function. We further discuss the prospects for UBM-Doppler analysis of alternative vertebrate systems increasingly studiedin developmental biology. The ability to correlate cardiovascular physi-ological phenotypes with their corresponding genotypes should lead tothe elucidation of mechanisms underlying normal development, as wellas embryonic disease and death.

cardiac morphogenesis; echocardiography; embryogenesis

THE ABILITY TO GENERATE transgene insertions andtargeted mutations in the mouse has led to newinsights into the genetic control of mammalian car-diovascular development. Among the earliest lessonsfrom mouse knockouts, most striking was the real-ization that the cardiovascular system is almostuniquely critical for embryonic survival: major de-fects in virtually all other organ and body systems,with the exception of the hematopoietic system, donot cause lethality in utero (4). Over the past decade,

a number of genetic factors and mechanisms under-lying cardiac development have been elucidated (e.g.,reviewed in Refs. 2 and 36), leading to increasinglyaccurate mouse models of human congenital heartdisease (e.g., reviewed in Refs. 5 and 8). Far lessunderstood are the precise roles of these geneticfactors in altering cardiovascular function, whichultimately causes disease and death. This void islargely due to the lack of effective noninvasive meth-ods to assess function in the developing mouse car-diovascular system, which also presents a majorlimitation in the analysis of disease progression inmouse models.

In this review, we discuss the recent advances inultrasound biomicroscopy (UBM)-Doppler echocardiog-raphy for analyzing cardiovascular function in the em-bryonic mouse, in utero, describing the range of phys-iological measurements that are possible using thisapproach. Particularly in the mouse, UBM-Dopplerhas distinct advantages over other imaging modalities,which will be discussed briefly to place the role ofUBM-Doppler in proper context. We further discussthe prospects for using UBM-Doppler in alternative

1This review article was based on work originally presented at the“NHLBI Symposium on Phenotyping: Mouse Cardiovascular Func-tion and Development” held at the Natcher Conference Center, NIH,Bethesda, MD, on October 10–11, 2002. Other review articles basedon presentations at this symposium have been published in a recentprevious release of Physiological Genomics, and more may be forth-coming.

Article published online before print. See web site for date ofpublication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: D. H.Turnbull, Skirball Institute of Biomolecular Medicine, New YorkUniv. School of Medicine, 540 First Ave., New York, NY 10016(E-mail: [email protected]).

Physiol Genomics 14: 3–15, 2003;10.1152/physiolgenomics.00008.2003.

1094-8341/03 $5.00 Copyright © 2003 the American Physiological Society 3

vertebrate systems commonly used for the study ofcardiac development.

BASIC PHYSIOLOGY OF THE EMBRYONICCARDIOVASCULAR SYSTEM

The embryonic mammalian cardiovascular systemcomprises the developing heart together with a num-ber of interdependent, complex, and rapidly chang-ing vascular networks, both within the embryoproper and in the extra-embryonic tissues that sup-port the embryo during normal in utero development(Fig. 1). In its most basic form, the cardiovascularsystem can be viewed as being composed of threemajor components: 1) the embryonic circulatory sys-tem, made up of the heart, and the embryonic arter-ies and veins, of which the aorta and cardinal veinare the main branches; 2) the yolk sac circulation,made up of the vitelline arteries and veins; and 3)the allantoic or umbilical circulation, of which theallantoic artery and vein are the main branches.Proper development and coordination of all threecirculatory systems are critical for normal cardiovas-cular function and embryonic survival. Indeed, anal-yses of a large number of mouse mutants indicatethat in utero lethality occurs in three distinct waves,corresponding to defects in the yolk sac (first wave),umbilical (second wave), and embryonic (third wave)circulatory systems (4). Therefore, any comprehen-sive approach to study functional development of themouse cardiovascular system should provide theability to measure physiological parameters in allthree of the developing circulatory systems over awide range of embryonic stages.

ANALYSIS OF EMBRYONIC CARDIOVASCULARFUNCTION: PREVIOUS TECHNIQUES

Analyzing cardiovascular function in the mouseembryo remains challenging, and relatively few suc-cesses have been reported (for a review, see Ref. 32).Most “classic” studies in developmental physiology

have employed either the servo-null micro-pressuresystem, refined by Clark and Hu (3), or implanted20-MHz pulsed Doppler ultrasound sensors, origi-nally developed by Hartley and Cole (14). Althoughthis latter approach has recently been applied tosurgically exposed mouse embryos (26, 27), most ofthe previous studies were done in the chick, becauseof easy access to the embryonic structures (for re-view, see Ref. 32). Such studies, however, are highlyinvasive and require significant perturbation andmanipulation of the developing embryo. Severalother imaging modalities are currently available,including high-speed videomicroscopy of surgicallyexposed mouse embryos (see Refs. 7, 26, 41); time-lapse videomicroscopy of cultured mouse embryos(see Ref. 24); magnetic resonance imaging of fixedmouse embryos (see Refs. 37, 38); high-speed fluo-rescent confocal videomicroscopy of zebrafish (seeRef. 17); and optical coherence tomography, recentlyapplied to chick embryos (see Ref. 50).

A number of echocardiographic techniques havebeen developed to study hemodynamics in the adultmouse, largely derived from human clinical ultra-sound (e.g., Refs. 16, 42, 49). However, spatial reso-lution in human echocardiographic systems, limitedto 300–500 �m at best, is marginal even for adultmice and is inadequate for mouse embryos. The fewstudies that have utilized human echocardiographysystems to examine mouse embryos have used real-time ultrasound images simply to locate the embryosin an anesthetized pregnant mouse, but have reliedon Doppler ultrasound to obtain blood velocity wave-forms as the basis for physiological analyses (13, 21,40). In late gestation mouse fetuses, it is possible toidentify separate structures such as the heart, aorta,umbilical vessels, and intracranial vessels and ob-tain Doppler signals from these different structures(28), but these stages are well beyond embryoniccardiac development. For the study of mouse modelsof normal and abnormal cardiovascular develop-

Fig. 1. Schematic of the embryonic mammalian car-diovascular system. The three major compartmentsare the embryonic circulatory system, which includesthe heart and intra-embryonic vascular bed (ACV,anterior cardinal vein; AoA, aortic arches; DAo, dor-sal aorta; HV, hepatic veins; ICA, internal carotidartery; PCV, posterior cardinal vein); the yolk sac orvitelline circulation (VA, vitelline artery; VV,vitelline vein); and the allantoic or umbilical circula-tion (AlA � allantoic, or umbilical, artery; AlV �allantoic, or umbilical, vein).

4 REVIEW: CARDIOVASCULAR UBM-DOPPLER

Physiol Genomics • VOL 14 • www.physiolgenomics.org

ment, a noninvasive high-resolution echocardiogra-phy system would seem to provide an ideal approachto obtaining physiological data (1).

UBM-DOPPLER: A NONINVASIVE, IN UTEROAPPROACH TO MEASURECARDIOVASCULAR FUNCTION

UBM systems, employing 40-MHz ultrasound toachieve spatial resolutions of 30 �m axial and 90 �mlateral, have been developed in our laboratory to imagethe embryonic mouse heart and cardiovascular systemnoninvasively (33, 39, 43, 44; reviewed in Ref. 45).UBM analysis of cardiovascular function in mouse em-bryos has been further extended by including 40–50MHz pulsed Doppler, enabling precise UBM image-guided Doppler measurements of blood velocity wave-forms (Fig. 2; Refs. 1, 23, 30). Recently, commercialinstrumentation, employing 19 to 55 MHz ultrasound(and with corresponding axial and lateral resolution asgood as 28 �m and 62 �m, respectively) has also be-come available to perform similar UBM-Doppler anal-ysis in the developing and maturing mouse cardiovas-cular system (10, 51).

UBM-Doppler differs from conventional clinicalechocardiography systems in the use of high-frequencyultrasound, which permits higher spatial resolution,although with poorer depth of penetration. The higher-frequency Doppler transducers also facilitate acquisi-tion of low-velocity blood flow signals. The two-dimen-sional UBM image is generated by mechanical motionof the imaging transducer (Fig. 2), so that the temporalresolution (frame rate) is limited by the sweep speed ofthe transducer motion, typically 8–10 frames/s (33,51). The pulsed-wave Doppler beam allows a specificsmall area (sample volume) to be interrogated; this

sample volume is guided by the UBM image itself (1,30, 51). The Doppler beam should be aligned with thedirection of blood flow as closely as possible, since theDoppler frequency shift and subsequent calculations offlow velocity vary as the cosine of the angle betweenthe flow direction and the Doppler beam. Mouse em-bryos may be imaged through the maternal abdomen(10, 30, 39, 51) or “semi-invasively,” whereby the em-bryos are pulled out through a small abdominal inci-sion but maintained in utero (23, 33). Because of theembryo’s extreme sensitivity to environmental pertur-bations and temperature, close thermoregulation of theentire maternal-embryonal unit is critical to obtainingphysiological data (23, 30).

UBM-Doppler provides a critical advantage overlower frequency (human) echocardiographic systems inits ability to interrogate separate circulatory beds inthe developing mouse embryo (30, 51). As describedbelow, UBM-Doppler has been used to investigate pa-rameters of cardiovascular function in both intra- andextra-embryonic circulatory systems from early embry-onic day 8.0 (E8.0) through E14.52 and in mouse fe-tuses through the end of gestation.

Intracardiac UBM-Doppler

UBM has been used to image the mouse heart fromembryonic (Fig. 3; Refs. 23, 39, 43, 44, 51) to neonatalstages (9, 29, 51). This has enabled noninvasive, in vivoassessment of function using indices derived from di-mensional analyses of the cardiac chambers (see be-low). UBM has also been used to guide 40-MHz Dopp-

2“E0.5” is defined as noon of the day a vaginal plug is detectedafter overnight mating.

Fig. 2. Schematic of the ultrasound biomicroscopy (UBM)-Doppler system (left) and a representative UBM imageof a stage E11.5 mouse embryo (right). A heating pad positioned under the pregnant mouse and warmedphysiological saline buffer (PBS) in the Petri dish help to maintain physiological temperatures (37 � 1°C) for boththe pregnant mouse and the embryos. The imaging and Doppler transducers are precisely calibrated so that theUBM image can guide the positioning of the Doppler sample volume. Since the Doppler sample volume dimensionis �1 mm or less, it can be seen that the different circulatory beds (intra-embryonic, vitelline/yolk sac, andumbilical/allantoic) may be imaged and analyzed separately. Scale bar (bottom right in UBM image) � 1 mm. Al,allantoic vessel; B, brain; P, placenta; U, uterine wall; V, vitelline vessel.

5REVIEW: CARDIOVASCULAR UBM-DOPPLER


ler measurements in the early embryonic mouse heart,which should allow analysis of diastolic function basedon intracardiac Doppler waveforms from the earlieststages of heart development (Fig. 3; Ref. 1).

Dorsal Aorta: A Window to EmbryonicCardiovascular Function

The dorsal aorta is easily identified with UBM fromapproximately E9.5 onward and can be interrogatedwith UBM-Doppler to measure aortic blood flow veloc-ity (Fig. 4; Refs. 1, 23, 30, 33). As the primary artery inthe developing mouse embryo, analysis of Dopplerwaveforms from the dorsal aorta has provided the basisfor our characterization of blood flow (33) and cardiacsystolic function in normal mouse embryos (30) and,more recently, in mutant mouse embryos with definedmorphological defects in cardiac development (31, 34).

Extra-Embryonic Circulations: Umbilical and YolkSac Blood Flow

From our earliest imaging studies in mouse embryos,the umbilical vessels were recognized as providing aneasily identifiable extra-embryonic circulatory system

that can be studied with UBM-Doppler (Fig. 5; Refs. 1,30, 39). Yolk sac vessels can also be visualized on UBM atmost embryonic stages (23, 39), and we have recentlydetermined that they are readily interrogated with Dopp-ler, although no systematic study of vitelline blood flowcharacteristics has been performed at this point (Fig. 5).

Secondary Embryonic Circulatory Systems: CerebralBlood Flow

Moving up the vascular tree from the major embry-onic and extra-embryonic circulatory systems, we havealso acquired Doppler waveforms from cerebral arte-rial vessels. The major cerebral arteries are evident onUBM as a result of the moving speckle patterns, andUBM-Doppler interrogation has yielded clear bloodvelocity waveforms, indicating that it should be possi-ble to investigate cerebral blood flow in developingmouse embryos (Fig. 6).

UBM-DOPPLER CHARACTERIZATION OFCARDIOVASCULAR FUNCTION

Cardiac output, perhaps the most fundamental mea-sure of cardiovascular function, may be defined simply

Fig. 3. Intracardiac UBM-Doppler. Left: the external relief of the heart (including the interatrial and interven-tricular grooves, and the atrioventricular groove) can be visualized readily by UBM, which can then guideintracardiac Doppler interrogation. Because the cardiac inflow and outflow axes are almost at 180°, inflow andoutflow signals are generated in opposite directions. Right: inflow Doppler data from E8.5 and E9.5 mouseembryonic hearts demonstrate the presence of separate E and A waves, with A wave dominance; increased inflowand outflow velocities are observed in the older embryo. Scale bar (bottom right of UBM image) � 1 mm. A � Awave of “active” ventricular filling due to atrial systole; E � E wave of “passive” ventricular filling duringventricular relaxation; LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle.



as the volume of blood pumped by the heart per unittime. Cardiac output is influenced primarily by fourfactors: heart rate, myocardial contractility, preload,and afterload (25). Contractility may be defined as themuscle’s intrinsic capacity to do work, independent ofthe loading conditions. However, the force a musclegenerates and its extent of shortening also depend onhow much it is stretched before initiation of contraction(preload) and the load against which it contracts (af-terload). Current ultrasound techniques do not permitprecise delineation of the contribution of each of theseparameters to the cardiac output. In addition, ultra-sound parameters of cardiac function are all influencedby loading conditions, and many are interdependent.Therefore, we prefer a multi-parameter approach toassessing cardiovascular function, which we believe isless susceptible to the limitations of any single UBM-Doppler measurement. Such an approach can yieldmuch information about cardiovascular physiology, in-cluding cardiac output and (systolic) cardiac work,myocardial mechanics, properties of the vascularbed(s), altered blood flow distribution, and character-ization of abnormal flow patterns linked with specificgenotypes and their morphological phenotypes.

UBM Indices of Cardiac Function

Despite some limitations (see below), UBM imagingcan provide useful hemodynamic information, notavailable from any alternative noninvasive imagingapproach. The changes in cardiac dimension duringcontraction may be used to gauge cardiac function (34,39), while the measurement of vessel size, in conjunc-tion with Doppler flow waveforms, allows calculation ofvolume flow (33).

Although it is difficult to obtain precise boundaries ofeach individual ventricle, we have been able to trace

the epicardial biventricular areas in embryos stagedE10.5–E14.5 in systole and diastole (34, 39). To over-come the poor temporal resolution, we currently recorddiastolic and systolic ventricular dimensions for atleast 10 consecutive cycles and use the maximal andminimal values to estimate the end-diastolic (EDA)and end-systolic (ESA) areas, respectively, and to de-rive the fractional area shortening [(EDA � ESA)/EDA]. In the unseptated embryonic heart, biventricu-lar fractional area shortening also serves as a surro-gate for cardiac ejection fraction. From end-diastolicbiventricular area, we can gauge whether there iscardiac dilatation, which should be observed in some,although not all, forms of congestive cardiac failure.

Finally, UBM can detect gross defects, such as peri-cardial effusions and general ascites (hydrops) of theembryo, which are signs of elevated central venouspressures and diastolic cardiac failure.

Doppler Indices of Cardiovascular Function

Doppler waveforms, acquired from the developingembryonic and extra-embryonic circulatory systems,provide a wealth of information pertinent to in uteromouse cardiovascular function (6, 13, 30, 35). In thissection, we outline the main parameters currentlymeasured with Doppler, and further discuss the func-tional significance of a number of these parameters insubsequent sections. Figure 7 provides schematicDoppler waveforms, defining the parameters discussedbelow.

Cycle length/heart rate. Cycle length (CL) is definedas the total time of one complete ejection cycle (inseconds), from the onset of one beat’s flow to the onsetof the next beat’s forward flow. Heart rate (HR) isderived simply from CL, as HR � 1/CL (in reciprocalseconds, s�1), or more commonly as HR � 60/CL (in

Fig. 4. UBM-Doppler of the dorsal aorta in a sagittal section. In our experience, the dorsal aorta can be alignedwith the Doppler beam to within 20° in most cases, an important consideration in obtaining the true blood flowvelocities within the aorta. As can be seen, the aortic diameter can also be measured, allowing for calculation ofcross-sectional area; this, in combination with the Doppler velocity, can be used to derive blood flow volume. Theslanted white lines (near the center of the UBM image) outline the approximate sample volume interrogated bypulsed-wave Doppler. Scale bar (bottom right of UBM image) � 1 mm. DAo, dorsal aorta; H, heart; S, spine; U,uterine wall.



beats/min). We have been able to discern clear Dopplersignals from mouse embryos as early as the 7-somite(early E8) stage (23), thus providing accurate heartrates from the onset of heart beating through mid-gestation (30).

Peak velocity. Peak velocity (PV) is the maximumdetected blood velocity (in mm/s).

Acceleration time/deceleration time. Accelerationtime (AT), also called rise time, is the time from onsetof systolic forward flow to peak velocity (in ms). Con-versely, deceleration time (DT) is the time from peakvelocity to the end of forward flow (in ms).

Ejection time/non-ejection time. Ejection time (ET)ET is the total time of systolic ejection, or forward flow:ET � AT � DT (in ms). Non-ejection time (NET) is thetime from the end of ejection to the onset of forwardflow of the next beat (in ms). ET may also be normal-ized to cycle length, as a measure of systolic work.

Velocity time integral. Velocity time integral (VTI) isthe area under the spectral Doppler flow velocity en-velope, when traced along its outer edge (in mm).

Pulsatility indices. Pulsatility indices are used, espe-cially in the characterization of umbilical flow dynam-ics, and in other vessels where the end-diastolic veloc-

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Fig. 5. Extra-embryonic circulations: umbilical/allantoic (top) and yolk sac/vitelline (bottom) blood flow in an E12.5mouse embryo. The slanted white lines (near the center of the UBM images) outline the approximate samplevolume interrogated by pulsed-wave Doppler. Vessels of the umbilical/allantoic and yolk sac/vitelline circulationscan be interrogated separately by UBM image-guided Doppler. Because the respective arteries and veins flow inopposite directions, their Doppler waveforms are distinct and appear on either side of the velocity baseline. TheDoppler waveforms show that, as expected at this stage, the umbilical circulation displays greater velocities dueto greater blood flow volume. Notably, diastolic arterial velocities in both beds drop to zero, a finding that indicatesrelatively high “downstream” vascular impedance. However, as gestation advanced, the progressive decrease inplacental vascular impedance was reflected in changes in not only the umbilical artery, but also the umbilical vein(30). Scale bar (bottom right of UBM images) � 1 mm. Al, allantoic vessel; Am, amniotic membrane; H, heart; P,placenta; L, an embryonic limb; U, uterine wall; V, vitelline vessel; Y, yolk sac.



ity may not be zero (i.e., may not return to baseline). Inthis case, the ratio of peak (systolic, S) to trough (dia-stolic, D) velocities, S/D, or the Pourcelot resistiveindex, (S � D)/S, is often used to quantify arterialwaveform pulsatility and indirectly provides informa-tion on “downstream” vascular impedance. Pulsatilityindices in umbilical vein (30) may also help character-ize “upstream” placental impedance; in principle, thesame concept may be applied to other vascular beds.

Isovolumic contraction time/isovolumic relaxationtime. Isovolumic contraction time (ICT) is the intervalbetween mitral valve closure and aortic valve opening(in ms), whereas isovolumic relaxation time (IRT) isthe time between aortic valve closure and mitral valveopening (in ms).

E/A ratio. Ventricular inflow waveforms exhibit acharacteristic biphasic shape, with the E wave (repre-senting ventricular diastole or “passive filling”) preced-ing the A wave (representing atrial systole, a latediastolic event, or “active filling”). The ratio of E/A peakvelocities is often used as an index of diastolic function.

TOWARD A SYSTEMATIC APPROACH TO ASSESSEMBRYONIC CARDIOVASCULAR FUNCTION

Parameters of cardiovascular function may be cate-gorized as measures of blood flow, cardiac systolic anddiastolic function, and vascular impedance, althoughwithin each of these categories, parameters may over-lap with other parameters in other categories. In thefollowing sections, we outline a number of functionalmeasurements that may be made with UBM-Doppler.

Blood Flow

Theoretically, flow within a vessel can be calculatedif one knows the cross-sectional area of the vessel andthe flow velocities within the tube. In human (6, 35)and adult mouse studies (16, 49), certain assumptionsare made about blood flow physics and flow character-istics. However, with far lower blood flow velocities in amuch smaller vasculature, the embryonic mouse maynot allow such assumptions. In a first-order approxi-mation model incorporating UBM-Doppler data, we

Fig. 6. UBM-Doppler imaging of the cerebral circulation. The schematic shows the general anatomy of the Circleof Willis, which is also evident in the UBM image. Doppler waveform characteristics change in different regionswithin the cerebral circulation, and at different ages. Scale bar (bottom right of UBM image) � 1 mm. ACA,anterior cerebral artery; ACom, anterior communicating artery; BA, basilar artery; ICA, internal carotid artery; lv,lateral ventricle; PCA, posterior cerebral artery; PCom, posterior communicating artery; P2, postnatal day 2; 3v,third ventricle; 4v, fourth ventricle.



recently concluded that the spatial velocity profile inthe mouse embryonic dorsal aorta is likely parabolic,not flat, with laminar flow. Using this model, we esti-mated that the E13.5–E14.5 mouse embryo generatesa dorsal aortic stroke volume of �0.50 mm3 (33). Fur-thermore, spectral Doppler analysis, such as thatfound in UBM-Doppler, appears preferable to analysis

using mean phasic Doppler, for calculation of volumet-ric flow, given the assumptions needed for the lattertechnique (33). Advances in our understanding of thephysics of blood flow in the developing embryo willrefine our ability to characterize volumetric blood flowin different vascular beds during cardiac development.

Cardiac Systolic Function: Work vs. Contractility

No ideal index of intrinsic cardiac contractility ex-ists. Investigators employing echocardiography, in-cluding UBM-Doppler, have typically used indices ofsystolic cardiac work as useful surrogates. Dopplerassessment of blood flow provides a reasonable assess-ment of overall ventricular function (16). Ejection timeand peak flow velocity have been shown to reflectsystolic work and parallel changes in contractilitywhen used serially in the same animal. Changes inflow acceleration (�velocity/�time) also correlate withchanges in contractility (16). As mentioned above, allsuch indices are influenced both by loading conditionsand by heart rate (16). Acceleration time has beentouted as an index of cardiac contractility (46, 47)independent of loading conditions (46). At present,however, it is not known how to normalize accelerationtime for changes in heart rate, which increases signif-icantly as gestation progresses (23, 30).

Cardiac systolic work and output increase geometri-cally between E9.5 and E14.5, by all UBM-guidedDoppler indices studied, including heart rate, peakaortic flow velocity, velocity-time integral, and ejectiontime as a proportion of the cycle length. Maturation ofcardiac contractility is less clear, however (30). In an-other study utilizing a conventional clinical ultrasoundsystem, isovolumic contraction time decreased frommid to late gestation, a finding attributed to increasingventricular pressures (13). However, isovolumic con-traction time is clearly lengthened by increasing after-load, is also dependent on preload (since strength ofcontraction depends on the preload), and is influencedby the heart rate.

Cardiac Diastolic Function

Diastole is a complex phenomenon that incorporatesactive ventricular relaxation, passive properties ofmyocardium, and contributions of atrial systole (11).The most commonly used index of diastolic function isthe ratio of the E and A waves across the atrioventric-ular inflow. Although neither is completely indepen-dent of the other, certain abnormal flow patterns typ-ically emerge as “diastolic dysfunction” (i.e., meaningworsening ventricular compliance, or increasing ven-tricular stiffness) progresses (for review, see Ref. 35).In animal work, a reversed E/A ratio or a monophasicA wave has been taken to indicate a stiff ventricle.Indeed, the progression of patterns in the maturingmouse embryo has been interpreted as support for anincreasingly compliant (less stiff) ventricle (13). UBM-Doppler studies of embryonic diastolic function areonly recently being undertaken, and preliminary re-sults in our laboratory and others (51) indicate that

Fig. 7. Schematics of Doppler waveforms in various cardiovascularcompartments. Top: representative of dorsal aortic or early to mid-gestation umbilical arterial flow, which typically exhibits a rapidupstroke and returns to zero velocity during diastole. AT, accelera-tion time; CL, cycle length; DT, deceleration time; ET, ejection time;NET, non-ejection time; PV, peak flow velocity; VTI � velocity-timeintegral, the area under the waveform. Middle: representative ofumbilical venous flow, which typically undulates with a non-zerovelocity; the pulsatility of the wave depends on many factors, includ-ing gestation age and “upstream” as well as “downstream” vascularimpedance. D � diastolic, or trough, velocity; S � systolic, or peak,velocity. Bottom: representative of intracardiac flow, showing atrio-ventricular inflow (“inflow”) in a direction opposite from ventriculo-arterial outflow (“outflow”). A � A wave of “active” ventricular fillingdue to atrial systole; E � E wave of “passive” ventricular filling dueto ventricular relaxation; ICT, isovolumic contraction time; IRT,isovolumic relaxation time.



biphasic inflow patterns exist from close to the earlieststages of heart development (Fig. 3).

Isovolumic relaxation time is sensitive to the rate ofventricular relaxation but because it is also influencedby aortic diastolic pressures and atrial pressures, isload dependent (16). It should be remembered thatcardiac valves do not develop until approximatelyE13.5 in the mouse embryo. Prior to this stage, regur-gitation of blood flow is prevented by the dynamicapposition of the endocardial cushions. We questionwhether changes in the timing of such cushion apposi-tion may occur independently of changes in functionalmyocardial maturation, potentially confounding theutility of Doppler diastolic indices.

Peripheral Vascular Impedance and BloodFlow Distribution

Using UBM-Doppler, it should be possible to de-rive volumetric flows and distribution of blood flow/cardiac output to various intra- and extra-embryonicvascular beds, which may change during normaldevelopment (18) and be altered in mouse mutantmodels. As ejection ends, Doppler parameters willreflect the “downstream” impedance, that is, theimpedance of the peripheral vascular bed distal tothe point of Doppler interrogation. Thus, for exam-ple, the deceleration and non-ejection times of theaortic waveform, and the ratio of the peak-troughumbilical arterial velocities are influenced by thecapacitance of their respective “downstream” vascu-lar beds. Venous flow (e.g., umbilical venous) mayalso provide some insights into “upstream” vascularbed impedance (30). In principle, these techniquesshould enable us to approximate the relative vas-cular impedances of the intra-embryonic bed, theplacenta, and the vitelline circulation/yolk sac bed(Fig. 5).

Data from the normal mid-gestation mouse embryostrongly suggested, as expected, a progressively highercapacitance and lower impedance of the placental bed(30). Notably, in a study of mouse embryonic umbilicalblood flow using invasive techniques, umbilical arterialflow was abolished when the ventricles were artificiallypaced (27). This finding suggests the intriguing possi-bility that, in addition to the embryonic circulation’sdependence on an optimum heart rate for efficientcardiac output, the distribution of the cardiac output isexquisitely sensitive to such changes in heart rate andcardiac output.

PUTTING IT ALL TOGETHER: ESTABLISHMENT OFEARLY CIRCULATION

When cardiac function commences and true circula-tion is established are fundamental questions in devel-opment. UBM-Doppler has allowed investigators torecord the earliest cardiac contractile and circulatoryevents in the in utero mouse embryo. Srinivasan et al.(39) and Zhou et al. (51) have demonstrated flow inE8.5 embryos, at approximately the heart tube stage,although these were not staged precisely. More recent

data from our laboratory have demonstrated rhythmiccardiac contractions as early as the 5-somite (straightheart tube) stage, with intra-embryonic blood flow ev-ident by 7–8 somites (23). Notably, the onset of cardiacactivity correlated precisely with entry of red bloodcells into the embryo proper. All indices of cardiacsystolic work increased dramatically through E10.5and aligned nicely with previous UBM-Doppler dataobtained from mid-gestation mouse embryos (30).These results, the first obtained in utero, indicate thatestablishment and early development of the circulationare precisely coordinated, bringing together cardiacactivity, an intact vascular circuit, and oxygen-carry-ing red cells to support the growing embryo (23).

ABNORMAL BLOOD FLOW PATTERNSAND PHYSIOLOGY

The ability to correlate a detailed cardiovascularphysiological phenotype with a given genotype is anexciting development in functional genomics. To date,however, only scant data have been reported in modelsof abnormal mouse cardiovascular development. Sev-eral investigators (13, 21, 28, 40) have utilized theDoppler capabilities found in conventional humanechocardiography systems. Gui and colleagues (13)demonstrated presumed atrioventricular regurgitationin mid-gestation trisomy 16 mouse embryos on Dopplerwaveform analysis. Huang et al. (21) and Sullivan etal. (40) detected increased blood flow velocities, pre-sumably in the pulmonary outflow, in mouse embryosexhibiting abnormal gap junctional communication, in-cluding the connexin 43-overexpressing transgenic(CMV43) and connexin 43 knockout mouse. Changes inother Doppler functional parameters were also ob-served. It should be noted, however, that in theseexperiments, precise localization of abnormal Dopplersignals to the presumed abnormal region was not pos-sible, given the large Doppler sample volume and poorspatial resolution of the ultrasound system. Maki et al.(28) demonstrated abnormal pulsatility with semilu-nar valve regurgitation in late gestation (E18.5) lox�/�

mouse fetuses, which have structural alterations inarterial walls leading to aortic aneurysms. Interest-ingly, lox�/� fetuses demonstrated normal-appearingcardiac structures and dimensions, despite Dopplerevidence of reduced cardiac output and right heartfailure. The ability to localize abnormal flows in thisstudy was possible only because of the size and gesta-tional age of the mouse fetuses, which were well be-yond cardiac developmental stages. Disturbances incardiac rhythm may also be detected with Doppler.Huang and Linask (22) found that tribromoethanolanesthesia could induce premature atrial contractionsin mid to late gestation mouse embryos. Prematureatrial contractions also seemed to be more prevalent inCMV43 mouse embryos (21). Despite limitations ofspatial resolution and localization of Doppler abnor-malities, these studies demonstrate the potential ofeven conventional clinical ultrasound systems, in stud-ies of the developing mouse.



In our laboratory, we have previously demonstratedthe ability of UBM to detect depressed ventricularfunction and pericardial effusions, indicative of heartfailure, in the embryonic-lethal VCAM-1 homozygousnull mutant mouse (39). In other experiments, we havealso noted arrhythmias, including bigeminal rhythmsand progressive bradycardia in dying embryos (CKLPhoon, O Aristizabal, RP Ji, and DH Turnbull, unpub-lished observations). Experiments are currently under-way on other models of abnormal cardiovascular devel-opment (31, 34). Detection of abnormal cardiovascularphysiology is but a first step; elucidation of the mech-anisms underlying embryonic disease and death willrequire a comprehensive and multi-parameter UBM-Doppler approach (32, 34).

ADDITIONAL COMPLICATING FACTORS

Despite the successes of UBM-Doppler in providingin vivo data relevant to cardiovascular function indeveloping mouse embryos, it must be realized thatthere are several potentially complicating factors in-volved in the acquisition of these data.

The low frame rate of currently available, mechani-cally scanned UBM systems (8–10 images/s), in thecontext of normal embryonic heart rates of 3–5 beats/s,limits its temporal resolution. This problem will onlybe overcome by the future development of higher framerate UBM systems, via the introduction of either fastermechanical scanners or high-frequency phased-arraytransducers (see Ref. 45, also http://www.visualsonics.com). Furthermore, the spatial resolution of UBM(e.g., 30 �m axial and 90 �m lateral resolution at 40MHz), while excellent compared with clinical ultra-sound systems, is still marginal for mouse embryowork, where, for example, the dorsal aorta diameter is�300 �m. Therefore, the ability to gauge preciselycardiac dimensions throughout the cardiac cycle issuboptimal. Moreover, at the high UBM-Doppler fre-quencies used (40–50 MHz), blood is echo-dense, andthe endocardial lumen (and hence myocardial wallthickness) cannot be visualized in the early to mid-gestation mouse embryo (39, 51). Interestingly, thisproblem appears to resolve somewhat in late gestation,so that it may be possible to characterize intracardiacanomalies later in gestation (51).

Anesthesia, although necessary to acquire reproduc-ible data, may affect physiological measurements. Theideal anesthetic agent and regimen for mouse cardio-vascular work have not been identified. However, re-cent data in early to mid-gestation mouse embryosshowed that embryonic and maternal heart rates didnot change significantly between the start and the endof experiments. These data indicate that heart rates,which are exquisitely sensitive to cardiovascular per-turbations, were not significantly altered by differ-ences in anesthesia level, which are expected to changesignificantly over the 1-h examination period, since themouse is typically deep under anesthesia at the start,and starting to regain consciousness toward the end ofthe examination (23). Further research will be re-

quired to determine the effects of various anestheticregimens on a number of UBM-Doppler parameters inthe developing embryo.

Several experiments in our laboratory currently em-ploy a “semi-invasive” approach, in which the intactuterus is surgically exteriorized into a bath of physio-logical saline (at 37 � 1°C) for UBM-Doppler, main-taining all maternal, embryonic, and extra-embryoniccirculatory systems intact (23, 31, 33, 34). This ap-proach has been adopted to enable easier acquisition ofhigh-quality UBM-Doppler data from entire litters ofembryos, both at very early developmental stages (23)and in the characterization of mutant phenotypes (31,34), although it has the obvious limitation of being lessamenable to longitudinal studies. Comparison of dataacquired noninvasively and semi-invasively showsthat, although the heart rates are slightly lower in theexteriorized embryos, other Doppler parameters arenot significantly different under the two conditions(23). Moreover, observed embryonic heart rates (bothsemi-invasive and noninvasive, and under anesthesia)are significantly higher than others reported in theliterature (23, 30), leading us to believe that these dataare closer to the physiological situation than those inprevious reports using more invasive measurementtechniques.

It should be noted that analytical techniques used inhuman (6, 35) and adult mouse (16) studies are notnecessarily applicable to the embryonic mouse. Contin-ual refinements in UBM-Doppler data analysis (30, 33)as well as technology (1, 10, 51) will be required forUBM-Doppler to fully realize its potential to shed in-sights into developmental cardiovascular physiology.

OTHER VERTEBRATE MODELS OFCARDIAC DEVELOPMENT

Although the mouse remains the preferred model ofmammalian development, other animal models havealso contributed to our understanding of cardiovascu-lar development and are potential candidates for UBM-Doppler imaging. For example, the chick embryo hasbeen a favorite vertebrate model for developmentalphysiologists because of its ease of access and ability tobe surgically manipulated and instrumented (see Ref.3). However, to our knowledge, the chick embryo hasyet to be studied by UBM-Doppler or other non-inva-sive ultrasound techniques. The exposed chick embryo,however, is amenable to optical imaging techniquesthat afford superb spatial and temporal resolution (50),so that the potential role of UBM-Doppler is not en-tirely clear.

With large-scale genetics screens available, thezebrafish has become a popular vertebrate modelwith which to identify and study genes involved incardiac development (12, 48). Evolutionary conser-vation means that much of zebrafish cardiac devel-opment is relevant to mammalian cardiac develop-ment, even though the fish heart is two-chambered.Notably, the early larval fish can survive throughdiffusion of oxygen alone, without the need for a



functional cardiovascular system; thus mutationsthat would lead to early embryonic lethality in themouse may still be characterized in early stage ze-brafish. Also, morphogenesis and function have beeneasier to study than in the mouse because of thetransparency of the young fish (12, 48). Cardiovas-cular function in both the developing and adult ze-brafish has been studied using high-speed videomi-croscopy and servo-null micropressure systems (12,19, 20, 48). Recently, Hove et al. (17), using high-speed fluorescent confocal in vivo imaging and sur-gical manipulation, demonstrated the important roleof shear forces in normal cardiac development. Todate, the use of Doppler ultrasound has been limitedto the study of adult zebrafish (15).

With the advantages of the zebrafish model in mind,we have begun to explore the feasibility of characterizingzebrafish cardiovascular hemodynamics using UBM-Doppler, especially in the developing heart. In prelimi-nary studies, we have been able to obtain Doppler signalsfrom the aorta at various stages from �48 h postfertil-ization through adulthood (Fig. 8; CKL Phoon, RP Ji, OAristizabal, D Yelon, and DH Turnbull, unpublished ob-servations), and at temperatures of 25–30°C. One majordifference between fish and mice is that fish are poikilo-thermal (cold-blooded), so that there is no single “physi-ological” temperature (15). One will therefore need toexercise caution in comparing cardiovascular data fromdifferent studies, since even small differences in temper-ature exert substantial influences on cardiac function(15, 32). In our hands, the consistent acquisition of Dopp-ler signals in zebrafish has been challenging; we believethat a small aorta, lower blood flow velocities, and thehigh reflectivity of fish skin/scales contribute to a lowsignal-to-noise ratio and suboptimal Doppler signals.Nevertheless, with continued refinement of techniques,the application of UBM-Doppler to the study of zebrafishmutations is likely to yield further insight into functional

genomics, complementing information derived fromother techniques (17, 19, 20) and that from the mouse.

SUMMARY AND FUTURE DIRECTIONS

UBM-Doppler has become an important technique inthe analysis and characterization of developmentalcardiovascular physiology. Challenges ahead in UBM-Doppler imaging include the development of higher-resolution (both spatial and temporal) technology, im-proved Doppler signal analysis, and advances in theunderstanding of microcirculatory flow physics. Inves-tigators are now beginning to move from largely de-scriptive studies of cardiovascular physiology to ad-dressing important developmental questions, in bothnormal and abnormal models. Insights into mecha-nisms of development, altered physiology, and embry-onic compensatory responses will ultimately necessi-tate the use of a variety of imaging modalities, inconjunction with invasive physiological assessmentand cellular biology techniques. Nevertheless, the dis-tinct advantages of UBM-Doppler as a non-invasiveimaging technology able to characterize mammalianembryonic hemodynamics assure it a central role in thefield of developmental cardiovascular physiology.

We thank our coworkers, Orlando Aristizabal and Rui Ping Ji, forcontributions in developing the methodologies described in this re-view, and Donald Christopher and Stuart Foster (University ofToronto) for providing unpublished details on the Doppler electronicsused in our laboratory prototype scanner. We also thank DebbieYelon and coworkers (Skirball Institute) for advice and help with thezebrafish measurements.

DISCLOSURES

The research described in this review has been supported in thepast by grants from the Whitaker Foundation and the AmericanHeart Association (AHA, New York City Affiliate) (to D. H. Turnbull)and by an AHA (Heritage Affiliate) Scientist Development Grant (toC. K. L. Phoon), and this research is currently supported by NationalInstitutes of Health Grants NS-38461 and HL-62177 (to D. H. Turn-bull) and HL-04414 (to C. K. L. Phoon).

Fig. 8. UBM-Doppler imaging of zebrafish. As can be seen, the zebrafish is much smaller in its larval stages thanthe developing mouse embryo. Here, we show arterial flow signals from the zebrafish aorta at 48 h postfertilization(48 hpf), whose velocities are far lower than are typical in the mouse embryo. The slanted white lines (near centerof the UBM image) outline the approximate sample volume interrogated by pulsed-wave Doppler. Scale bar (bottomright of UBM image) � 1 mm. Gelatin agar (G) is used to position and hold the zebrafish in place during imaging;H, head; P, petri dish bottom; T, tail.



D. H. Turnbull acknowledges that he has acted as a paid consul-tant to VisualSonics (Toronto, Ontario, Canada), a company makingUBM technology available to other researchers.

REFERENCES

1. Aristizabal O, Christopher DA, Foster FS, and TurnbullDH. 40 MHz echocardiography scanner for cardiovascular as-sessment of mouse embryos. Ultrasound Med Biol 24: 1407–1417, 1998.

2. Bruneau BG. Transcriptional regulation of vertebrate cardiacmorphogenesis. Circ Res 90: 509–519, 2002.

3. Clark EB and Hu N. Developmental hemodynamic changes inthe chick embryo from stage 18 to 27. Circ Res 51: 810–815,1982.

4. Copp AJ. Death before birth: clues from gene knockouts andmutations. Trends Genet 11: 87–93, 1995.

5. Dees E and Baldwin HS. New frontiers in molecular pediatriccardiology. Curr Opin Pediatr 14: 627–633, 2002.

6. DeGroff CG. Doppler echocardiography. Pediatr Cardiol 23:307–333, 2002.

7. Dyson E, Sucov HM, Kubalak SW, Schmid-Schonbein GW,DeLano FA, Evans RM, Ross J Jr, and Chien KR. Atrial-likephenotype is associated with embryonic ventricular failure inretinoid X receptor alpha �/� mice. Proc Natl Acad Sci USA 92:7386–7390, 1995.

8. Epstein JA. Developing models of DiGeorge syndrome. TrendsGenet 17: S13–S17, 2001.

9. Fatkin D, Christe ME, Aristizabal O, McConnell BK, Srini-vasan S, Schoen FJ, Seidman CE, Turnbull DH, and Seid-man JG. Neonatal cardiomyopathy in homozygous Arg403Gln �cardiac myosin heavy chain mutant mice. J Clin Invest 103:147–153, 1999.

10. Foster FS, Zhang MY, Zhou YQ, Liu G, Mehi J, Cherin E,Harasiewicz KA, Starkoski BG, Zan L, Knapik DA, andAdamson SL. A new ultrasound instrument for in vivo micro-imaging of mice. Ultrasound Med Biol 28: 1165–1172, 2002.

11. Gilbert JC and Glantz SA. Determinants of left ventricularfilling and of the diastolic pressure-volume relation. Circ Res 64:827–852, 1989.

12. Glickman NS and Yelon D. Cardiac development in zebrafish:coordination of form and function. Semin Cell Dev Biol 13:507–513, 2002.

13. Gui YH, Linask KK, Khowsathit P, and Huhta JC. Dopplerechocardiography of normal and abnormal embryonic mouseheart. Pediatr Res 40: 633–642, 1996.

14. Hartley CJ and Cole JS. An ultrasonic pulsed Doppler systemfor measuring blood flow in small vessels. J Appl Physiol 37:626–629, 1974.

15. Ho YL, Shau YW, Tsai HJ, Lin LC, Huang PJ, and HsiehFJ. Assessment of zebrafish cardiac performance using Dopplerechocardiography and power angiography. Ultrasound Med Biol28: 1137–1143, 2002.

16. Hoit BD and Walsh RA. In vivo echocardiographic assessmentof left ventricular function in transgenic and gene-targeted mice.Trends Cardiovasc Med 7: 129–134, 1997.

17. Hove JR, Koster RW, Forouhar AS, Acevedo-Bolton G,Fraser SE, and Gharib M. Intracardiac fluid forces are anessential epigenetic factor for embryonic cardiogenesis. Nature421: 172–177, 2003.

18. Hu N, Ngo TD, and Clark EB. Distribution of blood flowbetween embryo and vitelline bed in the stage 18, 21 and 24chick embryo. Cardiovasc Res 31: E127–E131, 1996.

19. Hu N, Sedmera D, Yost HJ, and Clark EB. Structure andfunction of the developing zebrafish heart. Anat Rec 260: 148–157, 2000.

20. Hu N, Yost HJ, and Clark EB. Cardiac morphology and bloodpressure in the adult zebrafish. Anat Rec 264: 1–12, 2001.

21. Huang GY, Wessels A, Smith BR, Linask KK, Ewart JL,and Lo CW. Alteration in connexin 43 gap junction gene dosageimpairs conotruncal heart development. Dev Biol 198: 32–44,1998.

22. Huang GY and Linask KK. Doppler echocardiographic analy-sis of effects of tribromoethanol anesthesia on cardiac function in

the mouse embryo: a comparison with pentobarbital. Lab AnimSci 48: 206–209, 1998.

23. Ji RP, Phoon CKL, Aristizabal O, McGrath K, Palis J, andTurnbull DH. Onset of cardiac function during early mouseembryogenesis coincides with entry of primitive erythroblastsinto the embryo proper. Circ Res 92: 133–135, 2003.

24. Jones EAV, Crotty D, Kulesa PM, Waters CW, Baron MH,Fraser SE, and Dickinson ME. Dynamic in vivo imaging ofpostimplantation mammalian embryos using whole embryo cul-ture. Genesis 34: 228–235, 2002.

25. Katz AM. Physiology of the Heart (3rd ed.). Philadelphia, PA:Lippincott Williams and Wilkins, 2001.

26. Keller BB, MacLennan MJ, Tinney JP, and Yoshigi M. Invivo assessment of cardiovascular dimensions and function inday �10.5 to �14.5 mouse embryos. Circ Res 79: 247–255, 1996.

27. MacLennan MJ and Keller BB. Umbilical arterial blood flowin the mouse embryo during development and following acutelyincreased heart rate. Ultrasound Med Biol 25: 361–370, 1999.

28. Maki JM, Rasanen J, Tikkanen H, Sormunen R, Makikal-lio K, Kivirikko KI, and Soininen R. Inactivation of the lysyloxidase gene lox leads to aortic aneurysms, cardiovascular dys-function, and perinatal death in mice. Circulation 106: 2503–2509, 2002.

29. McConnell BK, Jones KA, Fatkin D, Arroyo LH, Lee RT,Aristizabal O, Turnbull DH, Georgakopoulos D, Kass D,Bond M, Niimura H, Schoen FJ, Conner D, Fischman DA,Seidman CE, and Seidman JG. Dilated cardiomyopathy inhomozygous myosin-binding protein-C mutant mice. J Clin In-vest 104: 1235–1244, 1999.

30. Phoon CKL, Aristizabal O, and Turnbull DH. 40 MHz Dopp-ler characterization of umbilical and dorsal aortic blood flow inthe early mouse embryo. Ultrasound Med Biol 26: 1275–1283,2000.

31. Phoon CK, Aristizabal O, Zhou B, Wu B, Baldwin HS, andTurnbull DH. Abnormal cardiovascular physiology detected byin utero high-frequency Doppler precedes morphological defectsin NF-ATc1 deficient mouse embryos (Abstract). Circulation 102,Suppl: II109, 2000.

32. Phoon CKL. Circulatory physiology in the developing embryo.Curr Opin Pediatr 13: 456–464, 2001.

33. Phoon CK, Aristizabal O, and Turnbull DH. Spatial velocityprofile in mouse embryonic aorta and Doppler-derived volumet-ric flow: a preliminary model. Am J Physiol Heart Circ Physiol283: H908–H916, 2002. First published May 16, 2002; 10.1152/ajpheart.00869.2001.

34. Phoon CKL, Ji RP, Aristizabal O, Worrad DM, Wu B,Baldwin HS, and Turnbull DH. Complexity of embryonicheart failure: insights from NFATc-1 deficient mice (Abstract).Circulation 106, Suppl: II286–II2867, 2002.

35. Quinones MA, Otto CM, Stoddard M, Waggoner A, andZoghbi WA. Recommendations for quantification of Dopplerechocardiography: a report from the Doppler QuantificationTask Force of the Nomenclature and Standards Committee ofthe American Society of Echocardiography. J Am Soc Echocar-diogr 15: 167–184, 2002.

36. Rossant J. Mouse mutants and cardiac development: new mo-lecular insights into cardiogenesis. Circ Res 78: 349–353, 1996.

37. Smith BR, Johnson GA, Groman EV, and Linney E. Mag-netic resonance microscopy of mouse embryos. Proc Natl AcadSci USA 91: 3530–3533, 1994.

38. Smith BR. Magnetic resonance microscopy in cardiac develop-ment. Microsc Res Tech 52: 323–330, 2001.

39. Srinivasan S, Baldwin HS, Kwee L, Labow M, AristizabalO, Artman M, and Turnbull DH. Noninvasive, in utero imag-ing of mouse embryonic heart development with 40-MHz echo-cardiography. Circulation 98: 912–918, 1998.

40. Sullivan R, Huang GY, Meyer RA, Wessels A, Linask KK,and Lo CW. Heart malformations in transgenic mice exhibitingdominant negative inhibition of gap junctional communication inneural crest cells. Dev Biol 204: 224–234, 1998.

41. Tanaka N, Mao L, DeLano FA, Sentianin EM, Chien KR,Schmid-Schonbein GW, and Ross J Jr. Left ventricular vol-umes and function in the embryonic mouse heart. Am J PhysiolHeart Circ Physiol 273: H1368–H1376, 1997.



42. Tanaka N, Dalton N, Mao L, Rockman HA, Peterson KL,Gottshall KR, Hunter JJ, Chien KR, and Ross J Jr. Trans-thoracic echocardiography in models of cardiac disease in themouse. Circulation 94: 1109–1117, 1996.

43. Turnbull DH, Bloomfield TS, Baldwin HS, Foster FS, andJoyner AL. Ultrasound backscatter microscope analysis ofearly mouse embryonic brain development. Proc Natl Acad SciUSA 92: 2239–2243, 1995.

44. Turnbull DH. In utero ultrasound backscatter microscopy ofearly stage mouse embryos. Comput Med Imaging Graph 23:25–31, 1999.

45. Turnbull DH and Foster FS. Ultrasound biomicroscopy indevelopmental biology. Trends Biotechnol 20: S29–S33, 2002.

46. van Bel F, Schipper IB, Klautz RJ, Teitel DF, Steendijk P,and Baan J. Acceleration of blood flow velocity in the carotidartery and myocardial contractility in the newborn lamb. PediatrRes 30: 375–380, 1991.

47. Wallmeyer K, Wann LS, Sagar KB, Kalbfleisch J, andKlopfenstein HS. The influence of preload and heart rate on

Doppler echocardiographic indexes of left ventricular perfor-mance: comparison with invasive indexes in an experimentalpreparation. Circulation 74: 181–186, 1986.

48. Warren KS and Fishman MC. “Physiological genomics”: mu-tant screens in zebrafish. Am J Physiol Heart Circ Physiol 275:H1–H7, 1998.

49. Yang XP, Liu YH, Rhaleb NE, Kurihara N, Kim HE, andCarretero OA. Echocardiographic assessment of cardiac func-tion in conscious and anesthetized mice. Am J Physiol Heart CircPhysiol 277: H1967–H1974, 1999.

50. Yelbuz TM, Choma MA, Thrane L, Kirby ML, and Izatt JA.Optical coherence tomography: a new high-resolution imagingtechnique to study cardiac development in chick embryos. Cir-culation 106: 2771–2774, 2002.

51. Zhou YQ, Foster FS, Qu DW, Zhang M, Harasiewicz KA,and Adamson SL. Applications for multifrequency ultrasoundbiomicroscopy in mice from implantation to adulthood. PhysiolGenomics 10: 113–126, 2002. First published June 18, 2002;10.1152/physiolgenomics.00119.2001.



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