From the Skirball Institute of Biomolecular Medicine (S.S., O.A., D.H.T.)
and the Departments of Pediatrics (S.S., M.A.), Radiology (D.H.T.), and
Pathology (D.H.T.), New York University Medical Center, New York, NY; the
Joseph Stokes Research Institute (H.S.B.), Children's Hospital of
Philadelphia, Pa; and Roche Research Center (L.K., M.L.), Hoffmann-LaRoche
Inc, Nutley, NJ.
Correspondence to D.H. Turnbull, Skirball Institute of Biomolecular Medicine, New York University Medical Center, 540 First Ave, New York, NY 10016. E-mail turnbull{at}saturn.med.nyu.edu
Methods and Results—Transabdominal scans of mouse embryos staged
between 8.5 and 13.5 days of gestation (E8.5 to E13.5) were obtained in
anesthetized mice. Atrial and ventricular
contractions could be discerned from E9.5, and changes in cardiac
morphology were observed from E9.5 to E13.5. Hyperechoic streaming
patterns delineated flow through the umbilical, vitelline, and other
major blood vessels. Diastolic and systolic
ventricular areas were determined by planimetry of the
epicardial borders, and fractional area change was measured as an index
of contractile function. Significant increases in
ventricular size were documented at each stage between
E10.5 and E13.5, and the ability to perform serial imaging studies over
3 days of embryonic development is described. Finally, the detection of
vascular cell adhesion molecule 1 (VCAM-1) homozygous null mutant
embryos demonstrates the first example of noninvasive, in utero
analysis of cardiac structure and function in a targeted mouse
mutant.
Conclusions—We used 40-MHz echocardiography
to identify key elements of the early mouse embryonic
cardiovascular system and for noninvasive dimensional
analysis of developing cardiac ventricles. The ability to
perform serial measurements and to detect mutant embryos with cardiac
defects highlights the usefulness of the technique for investigating
normal and abnormal cardiovascular development.
High-frequency (40- to 50-MHz) ultrasound imaging, referred to as
UBM, allows high-resolution in utero imaging of live mouse
embryos.11 For this study, 40-MHz UBM was used to
image the developing hearts of live mouse embryos in utero from
gestational age 8.5 to 13.5 days (E8.5 to E13.5). We demonstrate 40-MHz
echocardiographic images of the early embryonic mouse
heart, allowing quantitative dimensional analysis of the
cardiac chambers and serial studies over several days of development.
In addition, we demonstrate the first noninvasive, in utero detection
of embryos homozygous for a single gene mutation (VCAM-1) resulting in
abnormal cardiac development and embryonic lethality. The ability to
monitor and follow cardiac development in utero by this approach should
be of great benefit in studying form and function relationships in
mouse models of cardiovascular disease.
The mouse was then placed on the lower level of a 2-level stage,
and a small water bath was fitted onto the skin to provide a coupling
medium for the transducer (Figure 1A
Image Acquisition
Imaging was started from one flank, and embryos were sequentially
imaged across the lower abdomen to the opposite flank. Embryos situated
too deep in the abdomen were scanned to document their presence but
were excluded from data analysis because of poor resolution.
The ability to scan adjacent embryos helped in tracking embryos;
however, in case of doubt, the embryos were skipped to avoid double
sampling, especially when associated with excessive spontaneous uterine
movements. Each litter had 6 to 10 embryos available for imaging
(average litter size of CD1 mice is 10 to 12 embryos; VCAM-1 mutant
mice typically have smaller litters, 5 to 8 embryos). After initial
localization of an embryo and optimal placement of the water bath,
sequential transabdominal linear scans were obtained at increments of
20 or 100 µm, depending on the size of the embryo and the region
of interest, thus imaging the entire embryo. Scan planes were modified
by changing the orientation of the mouse with respect to the scan
plane, and images were obtained in 2 orthogonal planes for each embryo.
An effort was made to obtain views approximating the transverse,
frontal, or sagittal planes but was sometimes limited to oblique planes
by the position of the uterus in the abdomen. A total of 234 embryos
staged between E8.5 and E13.5 were imaged with UBM over the course of
this study.
Toward the end of the present imaging study, a UBM
image-guided 40-MHz continuous-wave Doppler system was
developed15 15A and used to obtain nonambiguous in
utero heart rate data by measuring waveforms of the umbilical artery
and vein in 10 E13.5 embryos from 2 different litters (Figure 2
Image Analysis
Data are reported as mean±SD. To test for intraobserver variability,
10 embryos from E12.5 or E13.5 and 7 embryos from E10.5 or E11.5 were
analyzed. Four diastolic and 4 systolic
frames were selected for each embryo, and ventricular areas
were determined. Variability was expressed as difference from the mean
of the 2 results in percentage of the mean for diastolic
area, systolic area, and averaged FAC. The independent
t test was used to compare ventricular areas
between the groups.
As with clinical fetal ultrasound, embryonic orientation was first
determined by scanning from top to bottom. The consistent
orientation of normal mouse embryos within the uterus, with the
placenta and the tail to the right of the embryo, was confirmed and
used to identify cardiac structures. Cardiac contractions were
identified in some E8.5 embryos, and by E9.5 the common atrium and
ventricle could be distinguished. By E10.5 to E11.5, the distinction
between the presumptive LVs and RVs became more apparent. The outflow
tract was prominent (Figure 4A
Embryos have previously been imaged over 3 consecutive days, after
which the pregnancies went to term and no adverse effects on live-born
animals were observed.11 Although the objective
of the present study was to demonstrate the detailed anatomic data
on embryonic cardiac morphology possible with 40-MHz
echocardiography, we recognize the importance of
the technique for serial studies of individual embryos, particularly in
studies of mutant embryos to follow the dynamic progression of specific
cardiac defects. To further test the feasibility of serial studies, 4
litters were imaged on 3 consecutive days, from embryonic stage E11.5
to E13.5. After 1 additional day (E14.5), the anesthetized mothers were
killed by cervical dislocation and the embryos dissected and examined
under a dissection microscope. In all embryos in the 4 litters (n=31),
both the UBM images and gross morphology were normal.
Ventricular Dimensions and FAC
The intraobserver variabilities at E12.5 and 13.5 were 0.4±2.3% for
RV diastolic area, 1±1.6% for RV systolic area,
1.1±2.7% for RV FAC, 0.8±1.1% for LV diastolic area,
0.2±2.1% for LV systolic area, and 1.8±3.6% for LV FAC.
Corresponding values at E10.5 and E11.5 were 0.6±2.9% for combined
diastolic area, 0.6±0.7% for systolic area, and
0.7±7.3% for FAC. To study the reproducibility of data in different
litters and the variability of the technique, results from 2 litters at
E13.5 were compared (Table 2
Abnormal embryos observed during the course of this study included
numerous dead, resorbing embryos, which are expected and found in most
strains of mice.16 Two E13.5 embryos were noted
that showed features of hydrops, with evidence of pleural and large
pericardial effusions and a small, sluggishly contracting heart. In 1
of these animals, the calculated RV FAC was 19% and the LV FAC was
13%.
VCAM-1 Mutant Studies
Two VCAM-1 heterozygous mutant females were imaged at E10.5,
after which the mice were killed and their embryos dissected for
analysis. In the first mouse, 5 embryos were imaged on the left
uterine horn, 1 of which was identified as a mutant from the prominent
pericardial effusion, separation of allantois from chorion, and little
or no cardiac activity (Figure 6A
A third mouse had 8 embryos, which all appeared normal when imaged on 2
successive days (E10.5 and E11.5), and subsequent genotype
analysis showed that there were no homozygous mutants in the
litter. Finally, a fourth mouse had 7 embryos, which were imaged at
E12.5, 2 of which were obviously dead and resorbing, with a
characteristic echogenic rim around their hearts. Both dead embryos
were subsequently confirmed by genotype analysis to be
VCAM-1 homozygous mutants.
Video microscopy has been used previously to image the
heart in chick,18 19
rat,20 21 and mouse7 8
embryos. Unlike chick embryos, however, mammalian embryos must be
exposed by opening the maternal abdominal wall and uterus, or in the
case of early-stage embryos (E8.5 to E10.5), by maintaining the embryos
in culture, to allow optical imaging. In either case, viable embryos
can be maintained only for short periods of time, precluding the
possibility of serial studies over several days of development. MR
microscopy of embryonic mouse heart gives excellent
resolution,6 but noninvasive live cardiac MR
microimaging has not been demonstrated. The morphological changes in
the developing mouse heart as determined by 40-MHz
echocardiography are similar to those described
previously by scanning electron microscopy4 5 and
histological sections.2 There are
currently no competing in vivo imaging methods that combine the image
frame rate, resolution, and penetration of UBM for embryonic imaging
and serial in utero studies.
Ventricular epicardial area measures have previously
been correlated with ventricular mass and stroke volume in
chick embryos.18 With UBM, we obtained
ventricular epicardial areas and documented a significant
increase in ventricular size with gestational age, as has
been shown by more invasive methods.4 7 Given the
constant remodeling of the embryonic heart during cardiac
morphogenesis, calculation of ventricular volumes by use of
geometric assumptions is likely to be inaccurate; hence, we used FAC as
an index of contractile function. Moreover, during the earlier
embryonic stages (E10.5 to E11.5), although a prominent
interventricular sulcus is seen, the
interventricular septum is poorly
formed,5 and combined ventricular
area change was taken to be a more reliable estimate of function at
these stages. Ventricular areas measured by UBM at E12.5 to
E13.5 are comparable to those reported by Keller et
al,7 although our values for
ventricular areas were somewhat larger (eg, E13.5 LV/RV
diastolic area, 1.51±0.24/1.47±0.2
mm2 in the present study versus
0.96±0.05/1.19±0.04 mm2 in Keller et
al7 ). However, the 2 studies differ in the
methodology (noninvasive versus surgical exposure of embryos), the
projections used to obtain ventricular areas, and the
strains of mice imaged, all of which could contribute to the
differences noted.
One limitation of the current UBM imaging system is the
relatively slow frame rate (8 images per second). Given a heart rate of
120 to 200 bpm,7 9 this gives only 2 to 4 frames
for each cardiac cycle. We attempted to minimize the error by averaging
4 measures of apparent end systole and end diastole. The
rapid heart rates and the movements associated with maternal
respiration made it difficult to calculate heart rate visually from the
video images. However, the heart rates that we measured by Doppler
were similar to or even higher than those reported by other
investigators at similar embryonic stages. We noted a sensitivity of
the embryonic heart rates to the level of sedation and temperature and
the importance of minimizing heat loss.7 The
availability of UBM-guided Doppler, currently being developed in
our laboratory, will make it possible to monitor heart rate and blood
flow patterns while obtaining real-time images. The marked prominence
of the blood vessels, especially the umbilical vessels, which are
isolated from the rest of the embryo, makes the use of image-guided
Doppler interrogation an exciting possibility that would aid in
establishing functional and morphological correlation in the developing
mammalian cardiovascular system.
Ultimately, the value of the UBM imaging technology will be
revealed in future investigations of mutant mouse embryos with cardiac
defects. In the present study, VCAM-1 homozygous mutants were
identified in utero by 40-MHz echocardiographic
imaging. This is the first demonstration of the utility of UBM, or any
other imaging method, to noninvasively detect targeted mutant mouse
embryos with cardiac defects. Analysis of a limited number of
VCAM-1 heterozygous mutants demonstrated normal values of
ventricular FAC, providing further support for the
hypothesis that these animals do not have impaired cardiac function.
Future measurements of umbilical and cardiac Doppler waveforms in
VCAM-1 mutant embryos should clarify whether the cardiac-specific
phenotype is a secondary effect of abnormal placental
circulation13 or is a primary defect associated
with the loss of VCAM-1 expression.12 Serial
studies of normal and mutant embryos with cardiac defects provide a
unique method to determine the effects of specific genes on the
functional development of the cardiovascular
system.
Received January 13, 1998;
revision received March 20, 1998;
accepted March 26, 1998.
© 1998 American Heart Association, Inc.
Basic Science Reports
Noninvasive, In Utero Imaging of Mouse Embryonic Heart Development With 40-MHz Echocardiography
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—The increasing number of
transgenic and targeted mutant mice with embryonic cardiac defects has
resulted in the need for noninvasive techniques to examine cardiac
structure and function in early mouse embryos. We report the first use
of a novel 40-MHz ultrasound imaging system in the study of mouse
cardiac development in utero.
Key Words: ultrasonics • echocardiography • imaging • morphogenesis
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
The large
number of mouse models with congenital heart defects, many of which
result in early embryonic lethality,1 has
resulted in a need for new technologies that allow the study of cardiac
form and function in utero in mouse embryos. Previous investigations
have studied fetal mouse cardiac anatomy and vasculature by
examination of fixed specimens by histology,2 3
scanning electron microscopy,4 5 or magnetic
resonance microscopy.6 None of these methods have
provided real-time imaging of live embryos. More recent studies have
looked at mouse embryonic cardiac function with video
microscopy,7 8 which requires surgical exposure
of the embryos and is therefore unsuited to serial measurements.
Transabdominal 7.5-MHz Doppler9 has been used
to make noninvasive measurements of embryonic heart but was limited in
resolving anatomic details, particularly at earlier embryonic stages.
Echocardiography in the frequency range of 3 to 7.5
MHz is an established method for evaluating fetal cardiac
anatomy and function in humans beyond the first trimester and,
with transvaginal techniques, as early as late first
trimester.10 A higher-resolution, real-time
ultrasound imaging technique would be invaluable in the study of mutant
mouse phenotypes, especially during the early stages of cardiac
morphogenesis.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Animals
All animals used in these studies were maintained according to
protocols approved by the Institutional Animal Research and Care
Committee at New York University Medical Center. Timed pregnant CD1
mice (Charles River, Kingston, NY) and mice heterozygous for a null
mutation of VCAM-112 13 were anesthetized
with sodium pentobarbital (5 mg/100 g body wt IP) with added magnesium
sulfate (MgSO4 ·
7H2O, 10 mg/100 g body wt). Magnesium, a known
tocolytic agent, was added to decrease the spontaneous uterine
contractions that interfere with image acquisition. Although both
agents can have cardiac depressant actions that may introduce
confounding factors with functional assessments, nevertheless,
anesthesia is required to enable the acquisition of
reproducible data. In staging the embryos, day 0.5 (E0.5) was defined
as 12 noon of the day a vaginal plug was found after overnight mating.
Once the mouse was anesthetized, its lower abdomen and back
were wet-shaved to provide a clear window to the embryos, because hair
and the trapped microbubbles of air in the hair present an
impenetrable barrier to ultrasound at these high frequencies. 
and 1B). The stage holding the mouse was a simple wooden block
(LxWxH=85x120x40 mm) with a trough cut out (WxH=40x15
mm) in which the anesthetized mouse was laid. The water bath
was formed by either a 25-mm-diameter plastic cup or a Petri dish with
a 25-mm-diameter hole in the center pinned to the top level of the
stage to decrease the chances of extrinsic compression from a tight
bath. As has been reported previously,7 we noted
sensitivity of the embryos to temperature and attempted to maintain
normal maternal temperature with external heat lamps.
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Figure 1. Noninvasive in utero UBM imaging of mouse
embryonic heart. A, Photograph of experimental setup, including 2-level
stage (s), modified Petri dish (p), and imaging transducer (t). B,
Schematic showing details of imaging method. C, Example sagittal image
of E10.5 embryo showing heart (h) and dorsal aorta (da). ut indicates
uterus; nt, neural tube. Smallest scale increments on right are
100 µm.
A high-frequency (40- to 100-MHz) UBM system was developed and
constructed in our laboratory14 and was
previously demonstrated to be useful for in utero imaging of live mouse
embryos11 (Figure 1C
). For the present study,
a focused 40-MHz transducer was used, with measured lateral resolution
of 90 µm, axial resolution of 30 µm, and depth of
penetration of 7 to 10 mm.14 The mechanical
system scans over an 8-mm linear path, producing real-time images at a
rate of 8 images per second, which are then output as video to a
monitor/VCR for viewing and capture. Precise and repeatable control
over the position of the two-dimensional image plane was made possible
with a small motion stage and stepper motor, allowing the image plane
to be moved across the embryo in increments ranging between 10 and
100 µm. ).
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Figure 2. Forty-MHz Doppler evaluation of umbilical
vessels at E13.5. A, UBM image of placenta (pl) and umbilical vessels.
l, embryonic limb. Doppler sample volume is denoted by 2 parallel
lines. Smallest scale increments on right are 100 µm. B,
Doppler tracing showing arterial flow away from
transducer, represented as negative, and venous flow,
represented as positive. Heart rate is 180 bpm.
All imaging sessions were recorded in S-VHS video format
that allowed off-line review and analysis. Although cardiac
activity was discernible as early as E8.5, the heart structures at E8.5
to E9.5 were too small for reliable cardiac measurements to be made. In
the older embryos (E10.5 onward), scans through the embryonic heart
were selected and off-line analysis was performed with a
Hewlett Packard Sonos 1000 clinical ultrasound system. At E10.5 and
E11.5, embryos with scans in the transverse/oblique transverse plane
showing a small part of the atria and a view of both ventricles were
selected (Figure 3A through 3C). At E12.5
and E13.5, views representative of the 4-chamber view
were selected (Figure 3D through 3F). Calibration of measurement
software was performed by use of scale markers on the recorded
video screen. Sequential video fields were analyzed, and frames
representative of ventricular end systole
and end diastole were determined. Given the echogenicity of
the blood pool, the endocardial borders could not be identified; hence,
the ventricular area was determined by planimetry of the
epicardial borders (Figure 3A and 3D) similar to previous measurements
made with video microscopy on surgically exposed mouse
embryos.12 13 Because of the limitations of the
relatively low frame rate in reliably identifying end
diastole and end systole, an average of the maximal and
minimal areas obtained for 4 cardiac cycles was taken to be
representative. At E12.5 and E13.5, areas were
determined for RVs and LVs individually, whereas combined
ventricular area was determined at E10.5 and E11.5 because
of the lack of significant development of the
interventricular septum. The FAC was calculated for each of
these as the difference between the diastolic area and
systolic area, indexed as a percentage of diastolic
area.
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Figure 3. Transverse UBM scans at level of heart in E10.5 (A
through C) and E13.5 (D through F) embryos. A, Combined
ventricular diastolic area has been
planimetered from image shown in B. C, Systolic frame. Note
increase in atrial size during systole. D, Diastolic frame
at E13.5. RV and LV areas have been planimetered from image shown in E.
F, Systolic frame. Smallest scale increments on right of each
image (A through F) are 100 µm. Arrows denote embryonic axes in
this and subsequent figures; R, right; L, left; P, posterior; PS,
posterosuperior; a, amnion; la, left atrium; n, neural tube; p,
pericardium; pl, placenta; ra, right atrium; u, umbilical vessels; and
ut, uterus.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
UBM Images of Normal E8.5 to E13.5 Mouse Embryos
Images obtained by 40-MHz
echocardiography had some characteristics that
differ from lower-frequency clinical ultrasound. At this high
frequency, the blood pool appears highly echogenic, and as such, the
heart was readily identified on real-time images as a bright, highly
echogenic structure with rhythmic contractions (Figure 3). This also
imparted a dramatic speckle movement due to blood flow, which on real
time made the blood vessels prominent. This effect was less apparent on
still frames, in which the contrast from the moving speckle pattern was
lost. On real-time video images, the umbilical vessels (Figure 2A),
vitelline vessels, dorsal aorta (Figure 1C), great veins, and cerebral
vessels were all easily identified. through
4C), and 2 parallel streams of blood flow were apparent on real time.
Synchronous and discernible atrial and ventricular
contractions were apparent as early as E9.5, with atrial contraction
occurring at end diastole. At E12.5 to E13.5, there was a
marked increase in the size of all 4 chambers of the heart, and a
change in the configuration of the outflow tract was apparent (Figure 4D, E13.5). By sequential scanning, volumetric structures could be
followed through consecutive image planes (Figure 5). In stage E12.5 and E13.5 embryos, a
distinct "C"-shaped aorta could be traced, arising from the LV and
passing to the right of the pulmonary artery before looping to
the left (Figure 5C). The placenta changed from a discoid structure at
E10.5 to a larger planoconvex structure by E13.5, with prominent
sinusoids on its outer surface.
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Figure 4. UBM images trace development of outflow tract from
E9.5 to E13.5. Outflow tract before its division into pulmonary
artery and aorta is denoted by arrowheads in A through C. A, E9.5
embryo; primitive ventricle (lv) is prominent and bulbus cordis (bc)
continues into outflow tract. B, E10.5; a distinct rv component is
apparent. C, E11.5; outflow tract is larger but no separation is
evident. D, E13.5; transverse scan showing distinct pulmonary
artery (pa) and intertwining aorta (ao). Scale marker in D
represents 0.5 mm (500 µm). PS indicates
posterosuperior; a, amnion; la; left atrium; fl, forelimb; n, neural
tube; pl, placenta; ra, right atrium; and ut, uterus.
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Figure 5. Serial scans through heart in E12.5 embryo. Scans
are in oblique frontosagittal plane such that scan plane (A) is to
right and anterior compared with D, which is posterior and to left of
embryo. Difference in shapes of 2 ventricles is apparent, rv appearing
more triangular (A) compared with lv, which is more ellipsoid (C).
Distinct "C"-shaped aorta (ao) as it curves around
pulmonary artery (pa) is evident in C. Scale marker in D
represents 0.5 mm (500 µm). AI indicates
anteroinferior; dao, descending aorta; la, left atrium; li,
liver; lsc, left superior cardinal vein; p, pericardium; pl, placenta;
ra, right atrium; rsc, right superior cardinal vein; and s,
spine.
Ventricular area was obtained by planimetry of the
epicardial surfaces in a group of embryos (82 of 147 normal embryos
staged between E10.5 and E13.5) selected to have the best views for
performing quantitative analysis (see the Methods section).
There was a significant increase in ventricular size with
age. From E10.5 to E11.5, the combined ventricular area
increased from 1.47±0.33 to 2.02±0.21 mm2
in diastole and from 0.86±0.18 to 1.27±0.15
mm2 in systole (P<0.001). Similar
significant increases were noted from E12.5 to E13.5 for both
ventricles (Table 1). At these stages,
the RV and LV were well balanced, with no significant difference in
size. The FAC was 41±7% at E10.5 (n=15) and 37±5% at E11.5 (n=16).
The FAC was similar for both ventricles through E12.5 (RV, 32±7%; LV,
34±5%; n=21) and E13.5 (RV, 34±6%; LV, 34±6%; n=30) (Table 1).
Because combined ventricular area was used at the earlier
stages, no direct comparison was made with older embryos. At E13.5,
heart rate measured by 40-MHz Doppler was 196±27 bpm (Figure 2),
which is similar to reported values of 157±27
bpm9 and 173±7.5 bpm7 for
this gestational age.
View this table:
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Table 1. Ventricular Area and Fractional Area
Change for E12.5 and E13.5 Mouse Embryos ) and showed
no significant differences.
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Table 2. Variability in Ventricular Area Measures
for E13.5 Mouse Embryos
To investigate the utility of 40-MHz
echocardiography in the noninvasive detection of
targeted mouse mutant embryos with defects in cardiac development, we
performed in utero imaging studies on 4 litters (n=29) from VCAM-1
heterozygous intercrosses. VCAM-1 homozygous null mutant embryos have
been reported to die in utero, exhibiting 2 distinct
phenotypes.12 13 Approximately half of
the null mutants die before E11.5 and exhibit a failure of the
allantois to fuse with the chorion. The remaining VCAM-1 null mutants
appear to form a normal connection between allantois and chorion but
still die at E12.5 from cardiac-specific defects, including a lack of
epicardium and reduction of the ventricular
myocardium and intraventricular
septum. ;
compare with sagittal image of normal E10.5 embryo, Figure 1C). After
dissection, the embryo showed the same gross morphological features as
identified on ultrasound (Figure 6B) and was confirmed by Southern blot
analysis of yolk sac tissue to be a homozygous
mutant.12 In a second mouse, 9 embryos were
imaged and 2 identified as VCAM-1 null mutants, subsequently confirmed
by genotype analysis, with UBM features similar to
those noted above. The ventricular area measured in the
VCAM-1 homozygous mutant embryos (0.62 and 0.49
mm2) was well below that measured in normal E10.5
embryos (diastolic area, 1.47
mm2; systolic area, 0.86
mm2), and there was no discernible difference
between diastole and systole in the detected mutants.
Combined ventricular FAC was measured in 4 heterozygous
VCAM-1 mutants in the same litter and was found to have no significant
difference from FAC measured in normal E10.5 embryos (47±8% for
VCAM-1 heterozygotes versus 41±7% for normal embryos). Interestingly,
a third homozygous mutant was present in the litter that had normal
appearance on UBM images and also appeared normal when examined for
gross morphology.
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Figure 6. UBM detects VCAM-1 homozygous null mutant in vivo.
A, In utero sagittal UBM image of E10.5 VCAM-1 mutant demonstrating
unfused allantois (a), pericardial effusion (p), and ventricle (v).
Note normal appearance of neural tube. f indicates forebrain; m,
midbrain; h, hindbrain; c, chorion. Scale marker represents
0.5 mm (500 µm). B, Photograph of same embryo after
dissection.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
This study demonstrates the feasibility of noninvasive
real-time imaging of the early embryonic mouse heart in vivo by
high-frequency ultrasound. For the first time, dimensional
analysis of the cardiac ventricles has been demonstrated with
40-MHz echocardiography, and
ventricular area change has been measured as a noninvasive
index of contractile function. The developmental stages studied, E8.5
to E13.5, correspond to 
3 to 6 weeks of human
gestation2 17 and are the earliest hearts to be
studied in utero in the developing mouse. Gui et
al9 used transabdominal 7.5-MHz Doppler
ultrasound to obtain functional data from E10.5 to E19 mouse embryos,
but the frequency used was inadequate to image embryonic cardiac
anatomy, and therefore positioning of the Doppler sample
volume was only inferred from the similarity of the waveforms to those
measured in later-stage human fetuses. The future combination of
Doppler measurements, guided by UBM imaging to specific regions of
the developing heart, should provide a powerful method for assessing
cardiac function in mouse embryos. In this report, we provide
preliminary data to show the feasibility of in utero UBM-guided
Doppler measurements of umbilical blood velocity in E13.5 mouse
embryos.
Selected Abbreviations and Acronyms
E
=
embryonic gestational day
FAC
=
fractional area change
LV, lv
=
left ventricle, ventricular
RV, rv
=
right ventricle, ventricular
UBM
=
ultrasound backscatter microscopy
VCAM-1
=
vascular cell adhesion molecule 1
Acknowledgments
This research was supported by a grant from the Whitaker
Foundation. Dr Turnbull is an Investigator of the American Heart
Association/New York City Affiliate.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
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J. Liu, J. Du, C. Zhang, J. W. Walker, and X. Huang Progressive troponin I loss impairs cardiac relaxation and causes heart failure in mice Am J Physiol Heart Circ Physiol, August 1, 2007; 293(2): H1273 - H1281. [Abstract] [Full Text] [PDF]
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