imaging the embryonic heart: how low can we go? how fast can we get?

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Editorial Imaging the embryonic heart: How low can we go? How fast can we get? Anatomy is destiny. So I was told by a professor, but he was not referring to the many children who are born each year with structural heart defects. According to statistics available at the American Heart Association Web site (Americanheart.org), just less than 1% (8 in 1000) of infants are born with structural heart defects each year in the United States. This amounts to 40,000 children. In 1996, 4820 deaths due to congenital heart disease (CHD) were recorded. Due to improved surgical and medical care of infants and children with CHD, many will survive into adulthood. It is estimated that 1 million Americans are alive today with congenital structural heart defects. A number of experimental animal models have been used to attempt to understand how congenital heart defects arise. These models run the spectrum of the animal kingdom from those with the least to the most complex cardiac structures. The ability to manipulate single genes in the genome of the mouse has made the developing mouse the preferred model for which to study the role of single genes in the formation of the cardiac structures. Since an organism cannot survive without a heart, the congenital heart defects that infants are born with are predominately due to defects in the formation of the secondary structures of the heart, and not in the formation of the primary heart tube. The most common human congenital heart defects are in the formation of the atrial and ventricular septae, accounting for approximately one-half of all such defects. Other common defects include stenosis of the pulmonic and aortic valves, and the conotrun- cal defects, in which the connections of the right and left ventricles to the pulmonary artery and aorta, respectively, are abnormal. The conotruncal defects include complete trans- position of the great arteries, tetralogy of Fallot, double outlet right ventricle, and persistent truncus arteriosus. The beauty and complexity of the four chambered, dual circulation of advanced organisms has intrigued physicians and scientists for many years. How a primitive single heart tube is trans- formed into the complex heart of advanced organisms over a few days or weeks in utero (ontogeny recapitulating phylog- eny), and how this process may go wrong, has received intensive investigation over the past few decades. On page 217 in the current issue of the Journal of Molecu- lar and Cellular Cardiology, Bhattacharya and co-workers report on the effects of the deletion of the gene Cited2 on the development of the mouse heart. This study addresses a pressing need in this field of investigation; while elegant methods for creating heart defects through genetic manipu- lation are now routine, the ability to analyze the resultant dysmorphology has been limited. To put this problem into perspective, I like many expectant parents have marveled at the structural details observed with transcutaneous ultra- sonographic analysis of the early gestation human embryo. The heart of a comparably staged mouse embryo is several hundred-fold smaller, providing quite a challenge in terms of acquiring high-resolution images of the heart. Bhattacharya and co-workers attacked this problem with a magnetic reso- nance technique known as fast spoiled 3D gradient echo imaging with T1-weighting. Paraformaldehyde-fixed, agarose-embedded E15.5 mouse embryos in which the heart is approximately 1 mm in diameter were imaged. The agar- ose contained a solution of 2 mM gadolinium- diethylenetriamine pentaacetic anhydride (Gd-DTPA, a con- ventional MRI contrast agent) to improve water–tissue contrast. MRI signals were averaged four times to further improve signal-to-noise ratio and tissue contrast. Imaging was performed on an 11.7-T scanner, which uses a signifi- cantly stronger static magnetic field than the systems (at 1.5 T) available for general clinical purposes in humans. The final image resolution per pixel was on the order of 20 μm, however, for practical purposes, at least 3 pixels are needed to discriminate structures from one another, and thus the working resolution is on the order of 60–75 μm. The TIFF images were analyzed with commercially available software (Amira 2.3 TGS) and reconstructed into sagittal or coronal planes, or combined to generate 3D models of the heart. This analysis enabled the investigators to identify ASDs and VSDs as well as conotruncal and aortic arch defects in the embryos that lacked Cited2. The MRI analysis was compared to the gold standard, H+E-stained sections of these same hearts, and in a limited analysis (n = 6, unblinded) appeared nearly as sensitive, missing only a small (20 μm) VSD, which was below the limit of imaging resolution for this technique at this magnetic field strength. As with any new technology, a number of questions must be answered for its general utility in research to be discerned. How powerful is the method, and what is its cost- effectiveness and feasibility relative to established methods? What are the sensitivity, specificity and accuracy of the method? The traditional method for analyzing morphology Journal of Molecular and Cellular Cardiology 35 (2002) 141–143 www.elsevier.com/locate/yjmcc © 2003 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 2 8 2 8 ( 0 2 ) 0 0 3 0 6 - 1

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Page 1: Imaging the embryonic heart: How low can we go? How fast can we get?

Editorial

Imaging the embryonic heart: How low can we go?How fast can we get?

Anatomy is destiny. So I was told by a professor, but hewas not referring to the many children who are born eachyear with structural heart defects. According to statisticsavailable at the American Heart Association Web site(Americanheart.org), just less than 1% (8 in 1000) of infantsare born with structural heart defects each year in the UnitedStates. This amounts to 40,000 children. In 1996, 4820deaths due to congenital heart disease (CHD) were recorded.Due to improved surgical and medical care of infants andchildren with CHD, many will survive into adulthood. It isestimated that 1 million Americans are alive today withcongenital structural heart defects.

A number of experimental animal models have been usedto attempt to understand how congenital heart defects arise.These models run the spectrum of the animal kingdom fromthose with the least to the most complex cardiac structures.The ability to manipulate single genes in the genome of themouse has made the developing mouse the preferred modelfor which to study the role of single genes in the formation ofthe cardiac structures. Since an organism cannot survivewithout a heart, the congenital heart defects that infants areborn with are predominately due to defects in the formationof the secondary structures of the heart, and not in theformation of the primary heart tube. The most commonhuman congenital heart defects are in the formation of theatrial and ventricular septae, accounting for approximatelyone-half of all such defects. Other common defects includestenosis of the pulmonic and aortic valves, and the conotrun-cal defects, in which the connections of the right and leftventricles to the pulmonary artery and aorta, respectively, areabnormal. The conotruncal defects include complete trans-position of the great arteries, tetralogy of Fallot, double outletright ventricle, and persistent truncus arteriosus. The beautyand complexity of the four chambered, dual circulation ofadvanced organisms has intrigued physicians and scientistsfor many years. How a primitive single heart tube is trans-formed into the complex heart of advanced organisms over afew days or weeks in utero (ontogeny recapitulating phylog-eny), and how this process may go wrong, has receivedintensive investigation over the past few decades.

On page 217 in the current issue of the Journal of Molecu-lar and Cellular Cardiology, Bhattacharya and co-workersreport on the effects of the deletion of the gene Cited2 on thedevelopment of the mouse heart. This study addresses a

pressing need in this field of investigation; while elegantmethods for creating heart defects through genetic manipu-lation are now routine, the ability to analyze the resultantdysmorphology has been limited. To put this problem intoperspective, I like many expectant parents have marveled atthe structural details observed with transcutaneous ultra-sonographic analysis of the early gestation human embryo.The heart of a comparably staged mouse embryo is severalhundred-fold smaller, providing quite a challenge in terms ofacquiring high-resolution images of the heart. Bhattacharyaand co-workers attacked this problem with a magnetic reso-nance technique known as fast spoiled 3D gradient echoimaging with T1-weighting. Paraformaldehyde-fixed,agarose-embedded E15.5 mouse embryos in which the heartis approximately 1 mm in diameter were imaged. The agar-ose contained a solution of 2 mM gadolinium-diethylenetriamine pentaacetic anhydride (Gd-DTPA, a con-ventional MRI contrast agent) to improve water–tissuecontrast. MRI signals were averaged four times to furtherimprove signal-to-noise ratio and tissue contrast. Imagingwas performed on an 11.7-T scanner, which uses a signifi-cantly stronger static magnetic field than the systems (at1.5 T) available for general clinical purposes in humans. Thefinal image resolution per pixel was on the order of 20 µm,however, for practical purposes, at least 3 pixels are neededto discriminate structures from one another, and thus theworking resolution is on the order of 60–75 µm. The TIFFimages were analyzed with commercially available software(Amira 2.3 TGS) and reconstructed into sagittal or coronalplanes, or combined to generate 3D models of the heart. Thisanalysis enabled the investigators to identify ASDs andVSDs as well as conotruncal and aortic arch defects in theembryos that lacked Cited2. The MRI analysis was comparedto the gold standard, H+E-stained sections of these samehearts, and in a limited analysis (n = 6, unblinded) appearednearly as sensitive, missing only a small (20 µm) VSD, whichwas below the limit of imaging resolution for this techniqueat this magnetic field strength.

As with any new technology, a number of questions mustbe answered for its general utility in research to be discerned.How powerful is the method, and what is its cost-effectiveness and feasibility relative to established methods?What are the sensitivity, specificity and accuracy of themethod? The traditional method for analyzing morphology

Journal of Molecular and Cellular Cardiology 35 (2002) 141–143

www.elsevier.com/locate/yjmcc

© 2003 Elsevier Science Ltd. All rights reserved.PII: S 0 0 2 2 - 2 8 2 8 ( 0 2 ) 0 0 3 0 6 - 1

Page 2: Imaging the embryonic heart: How low can we go? How fast can we get?

involves the simple observation of the heart in whole mountfollowed by gross dissections or serial sectioning and stain-ing. A number of casting materials are available that preservethe architecture of the heart and facilitate detailed analysis.While this is very cost-effective and can easily identify majordefects, it has a number of limitations. The most criticallimitation, as anyone who has tried may attest to, is that dueto the small size of the embryonic mouse heart, gross dissec-tion is very difficult, so that defects inside the heart aredifficult to assess. Serial sectioning of the heart is used forthis purpose, but once the heart is sectioned critical anatomicrelationships that may only be evident in 3 dimensions arelost. Furthermore, sections that are not exactly oriented in aspecific plane may lead to spurious conclusions about ana-tomic relationships. Finally, this method is not applicable invivo, so that (1) fixation artifacts may arise, (2) functionalinformation is lost and (3) serial analyses of heart develop-ment in a single embryo are not possible.

For these reasons, a number of investigators have experi-mented with newer imaging modalities for assessing heartdevelopment. These methods can be broadly classified intomagnetic resonance based, ultrasonographic, and lightbased. The use of MR microscopy to evaluate embryonicmouse heart morphology was pioneered by Dr. BradleySmith and colleagues in 1994 (reviewed in Ref. [1]). Thestudy by Bhattacharya and co-workers in this issue of theJournal of Molecular and Cellular Cardiology has im-proved the technique with several modifications. Like theprior study, a high energy magnet (11.7 T) was used to imagefixed embryos. In each instance, spin echo imaging, a “brightwater” technique that makes proton-rich moieties (water, fat)look bright on the image (T1-weighted), was used. A majorimprovement appears to be the use of the fast 3D spoiledgradient images with T1-weighting used in the more recentstudy. This gives the image advantages of a T1-weightedsequence, while more rapidly acquiring 3D data using a moreefficient technique.

The images of the embryos in the current study in Journalof Molecular and Cellular Cardiology are remarkable. Thelevel of detail is extraordinary and underscores the high-resolution capabilities of the technique. The ability to editand reformat the images in different planes, and to view 3Dimages of the heart with readily available computer software,would be of great benefit to anyone who has tried to masterthe complex anatomic relationships of the normal or defec-tive embryonic heart. So how feasible is this method and howdoes it compare with other available methods?

One must obviously have access to a high field MR systemfor this analysis. While MR imaging machines are availablefor clinical use in many centers, the lesser field strength andhigh demand make these systems impractical for small ani-mal studies. The current high cost of this high field equip-ment would seem to preclude it becoming common to re-search laboratories except perhaps at major medical centers.A second concern is the technical expertise and time requiredfor imaging. In the current report the rate of successful

imaging is not reported, nor is a likely learning curve de-scribed. An important innovation is the use of the fast spoiled3D gradient echo sequence that reduced imaging time from astandard 36 h to approximately 9 h with little hands-on timerequired. While this certainly represents an improvement, ithardly seems that 9 h of imaging for each embryo wouldallow for high throughput analyses of mutant mice, andindeed only six mouse embryos were examined with thismethod. However, newer MR techniques have been devel-oped which will further decrease imaging time and improveimage quality. By contrast enhancing the cardiovascularstructures themselves, rather than just the agarose gel (as inthe current study), an even greater signal-to-noise ratio maybe obtained. Smith et al. [2] have employed selective umbili-cal vein cannulation and perfusion of the embryo with apredominantly intravascular contrast agent (BSA-DTPA-Gd). This approach results in a significant improvement indetecting vessels and other perfused structures from the sur-rounding soft tissues and reduces imaging time to approxi-mately 2 h per mouse, in part by decreasing the number oftimes the MRI signal must be averaged. The spatial resolu-tion using this approach is approximately 20 µm as well, butemploys a smaller coil and field of view. The contrast-enhanced techniques also permit more reliable results fromthresholding and segmenting tools in 3D image-processingmethods, and make serial quantitative analyses of tissues andchamber volumes more practical. At a recent NIH Sympo-sium on Phenotyping (Mouse Cardiovascular Function andDevelopment, October 10–11, 2002), RM Henkelman et al.reported on new methods to image up to 16 anesthetizedadult mice simultaneously in a single scanner, using indi-vidual coils for each mouse and multiplexing techniques.One can envision potentially screening multiple litters ofmouse embryos using this multiple coil approach should itscale down to the required level. Improved spatial resolutionat greater field strengths (17.6 T, A. Haase et al.) has pro-duced data suggesting that even myofiber orientation may bedetectable noninvasively.

Other methods for imaging embryonic mouse hearts arealso being developed. Transcutaneous ultrasonography usingstandard transducers (7–15 MHz) generally does not haveadequate resolution to define the fine structures. High-frequency (40 MHz) ultrasound has been used with somesuccess, with the advantage over MR being its ability toserially interrogate the heart over many days of developmentin utero. Limitations of this technique include the relativelyslow frame rate (8 s) compared to the heart rate requiring theaveraging of images, and the lesser quality of the staticimages as compared to MR. A third relatively new method isepiscopic fluorescence image capture, in which images ofsections are captured and reconstructed into 3D. This pro-vides high-resolution images, but can only be performed onfixed images and is very labor intensive. Confocal scanningmicroscopy provides exquisite detail of fixed embryonic or-gans but requires a fluorescent tag on the tissue. Micro-computed tomography and micro-positron emission tomog-

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Page 3: Imaging the embryonic heart: How low can we go? How fast can we get?

raphy techniques are in development for phenotypic andgene expression characterization, but have limitations in do-simetry and spatial resolution.

The true test of any new technique is the new informationthat it may provide. So what did we know about Cited2 andwhat have we learned? Cited2 (CBP/p300-interacting trans-activators with glutamic acid (E) and aspartic acid (D)-richtail) is a recently identified nuclear protein that binds withhigh affinity to the first cysteine–histidine-rich (CH1) regionof p300 and CREB-binding protein (CBP). The exact func-tion of Cited2 is not known; it has been proposed to functionas a co-activator for the AP-2 transcription factor, as well asan antagonist to the hypoxia inducible factor HIF-1a, bycompeting for binding to CBP/p300. HIF-1a mediates manyof the transcriptional responses to hypoxia, but like manytranscription factors, requires the CBP/p300 co-activator foractivity. Several laboratories have reported the effect of thetargeted deletion of the Cited2 gene, including a prior reportby Bhattacharya and co-workers [3–5]. Using whole mountobservations as well as H+E-stained sections these investiga-tors observed atrial and ventricular septal defects, conotrun-cal defects that included DORV and PTA, and aortic archmalformations. It is thus evident that these methods in thehands of experienced investigators can yield reliable analysisof embryonic heart dysmorphology.

It is encouraging that three different groups that indepen-dently derived Cited2 deleted mice observed a similar spec-trum of cardiac defects, in addition to neural tube and otherdefects. However, as evident in this and other studies, there issignificant variability in the phenotype even within a litter.This phenotypic variability is common in these types ofexperiments and presumably arises from polymorphisms atother genetic loci as well as perhaps unique adaptations to thegenetic insult. Extensive breeding of mice will be required tosort out the factors that influence the phenotype, and this issurely the place where a higher throughput imaging modalitywould have a great impact. Noninvasive evaluation of geneexpression using MRI methods may also prove useful inthese analyses. Localization of gene products, or serial evalu-ation of gene expression, may be possible using receptor-specific (targeted) MRI contrast agents. Two recent reportsdescribe the technique of targeting MRI contrast agents to atumor overexpressing a transferrin receptor [6], and agentsthat are activated intracellularly by b-galactosidase [7]. This

approach may permit more rapid genotypic as well as pheno-typic screening of embryos in utero. Using new 3D visualiza-tion tools and recent strategies to promote greater through-put, a more comprehensive evaluation of cardiacdevelopment may soon be possible.

References

[1] Smith BR, Johnson GA, Groman EV, Linney E. Magnetic resonancemicroscopy of mouse embryos. Proc Natl Acad Sci USA 1994;91:3530–3.

[2] Smith BR. Magnetic resonance microscopy in cardiac development.Microsc Res Tech 2001;52:323–30.

[3] Yin Z, Haynie J, Yang X, Han B, Kiatchoosakun S, Restivo J, Yuan S,Prabhakar NR, Herrup K, Conlon RA, Hoit BD, Watanabe M,Yang YC. The essential role of Cited2, a negative regulator for HIF-1alpha, in heart development and neurulation. Proc Natl Acad SciUSA 2002;99:10488–93.

[4] Bamforth SD, Braganca J, Eloranta JJ, Murdoch JN, Marques FI,Kranc KR, Farza H, Henderson DJ, Hurst HC, Bhattacharya S. Car-diac malformations, adrenal agenesis, neural crest defects and exen-cephaly in mice lacking Cited2, a new Tfap2 co-activator. Nat Genet2001;29:469–74.

[5] Weninger WJ, Mohun T. Phenotyping transgenic embryos: a rapid3-D screening method based on episcopic fluorescence image captur-ing. Nat Genet 2002;30:59–65.

[6] Weissleder R, Moore A, Mahmood U, Bhorade R, Benveniste H,Chiocca EA, Basilion JP. In vivo magnetic resonance imaging oftransgene expression. Nat Med 2000;6:351–5.

[7] Louie AY, Huber MM, Ahrens ET, Rothbacher U, Moats R,Jacobs RE, Fraser SE, Meade TJ. In vivo visualization of gene expres-sion using magnetic resonance imaging. Nat Biotechnol 2000;18:321–5.

Victor A. Ferrari, Steven A. Fisher *Divisions of Cardiology, University of Pennsylvania,

Pennsylvania, USA and Departments of Medicine andPhysiology, Case Western Reserve University,

422 Biomedical Research Building, 11100 Euclid Avenue,Cleveland, OH 44106-4958, USA

E-mail address : [email protected]

Received 5 November 2002; accepted 6 November 2002

* Corresponding author.Present address: Departments of Medicine and Physiology,

Case Western Reserve University,422 Biomedical Research Building, 11100 Euclid Avenue,

Cleveland, OH 44106-4958, USA.

143Editorial