In vivo ultrasound biomicroscopy in developmental biology

Transcription

1 In vivo ultrasound biomicroscopy in developmental biology Daniel H. Turnbull and F. Stuart Foster The mouse is the preferred animal model for studying mammalian developmental genetics and many human diseases. Ultrasound biomicroscopy (UBM) provides a unique real-time, in vivo micro-imaging approach for analyzing the phenotypes of mutant and transgenic mice from embryonic to adult stages. We review recent advances in the use of UBM and high-frequency ultrasound Doppler for analyzing the developing mouse cardiovascular system, and the application of in utero UBM image-guided injections for direct manipulation of mouse embryos. We describe currently available instrumentation, summarize the strengths and limitations of UBM and discuss the potential for future improvements to the technology. The growing need for in vivo micro-imaging technologies is now evident across a broad spectrum of biomedical research. Much of the recent research into in vivo microimaging approaches has been driven by the collection of increasingly accurate genomic and gene-expression data that have raised numerous questions about how genetic variations and mutations relate to development and disease. Current analysis of genotype phenotype relationships relies on the evaluation of large numbers of mutations in animal systems. The mouse is the most widely used mammalian model for such work but rapid phenotype analysis using the traditional tools of optical microscopy and histology has been difficult. The allure of in vivo micro-imaging derives from its potential to provide longitudinal structural and functional information on development and disease in individual mice noninvasively. The role of ultrasound in the milieu of mouse microimaging and phenotype analysis closely parallels its current role in clinical imaging. That is, the strengths of ultrasound in cardiology, obstetric, vascular and abdominal imaging appear most likely to extend to the mouse when the technology is scaled to achieve high-resolution and a level of functionality similar to that available with current clinical ultrasound systems. In addition, the real-time nature of ultrasound has led to its application in image-guided injection systems, enabling mouse embryos to be directly manipulated in utero. In this short review, we examine how far ultrasound has come in mouse phenotype analysis and image-guided manipulations, focusing on the role of high-frequency ultrasound imaging in studying disease and normal development in the mouse. Ultrasound biomicroscopy Ultrasound backscatter-microscopy or biomicroscopy (UBM) is a high-frequency ( MHz), pulse-echo ultrasound approach for imaging living biological tissues [1] with near-microscopic resolution (reviewed in [2]). In 1995, Turnbull et al. first reported the use of UBM to image mouse embryos in utero and to analyze a neural tube defect in a mutant mouse embryo [3]. Since then, improvements in the technology have permitted numerous investigations in the mouse.these investigations have been performed largely with prototype instruments [4 6], or with modified commercial scanners, which were originally designed for human ophthalmic imaging. More recently, a commercial UBM system designed specifically for mouse imaging and Doppler measurement of blood velocity has become available [7] (VisualSonics, Toronto Canada). As reviewed below, UBM in the frequency range MHz has been used for both 2D imaging and Doppler analysis in mouse embryos and neonates (Fig. 1). At these frequencies, spatial resolution in the µm range has been achieved, the resolution scaling linearly with frequency if the focusing parameters are fixed [2]. Ultrasound penetration in soft tissues is limited to 5 10 mm at these high frequencies, but this is sufficient for many microimaging applications in the mouse, and the corresponding increase in resolution (to µm) is necessary to visualize small anatomical structures, especially at embryonic and early postnatal stages. Echocardiography of the mouse cardiovascular system Echocardiographic assessment of the heart from fetus to adult is an important application of clinical ultrasound. Ultrasound imaging using diagnostic ultrasound instrumentation operating in the MHz frequency range has been successfully applied to a variety of mouse models of adult cardiovascular disease (reviewed in [8]). In these systems, optimized spatial resolution is µm, which is consistent with the resolution achieved in pediatric patient imaging. Even with this comparatively coarse resolution, it has been possible to examine, with some success, transgenic mouse models of cardiac hypertrophy [9], Daniel H. Turnbull Skirball Institute of Biomolecular Medicine, and Depts of Radiology and Pathology, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA. turnbull@saturn.med.nyu.edu F. Stuart Foster Sunnybrook and Women s College Health Sciences Centre, Mouse Imaging Centre at the Hospital for Sick Children, and Dept of Medical Biophysics, University of Toronto, 2075 Bayview Avenue, Toronto, Ontario, Canada M4N 3M5. stuart.foster@ swchsc.on.ca /02/$ see front matter 2002 Elsevier Science Ltd. All rights reserved. PII: S (02) S29

2 Review A TRENDS Guide to Imaging Technologies Trends in Biotechnology Vol. 20 No. 8 (Suppl.), 2002 Ultrasound transducer Injection microcapillary UBM electronics Imaging transducer Doppler transducer HF Doppler electronics Sterile PBS Image plane Cell suspension Modified petri dish Pins to secure petri dish to holding stage Modified petri dish UBM image plane Doppler beam Water Shaved skin Embryo Uterus Imaging beam Rubber membrane Ultrasound beam Uterine horn Cross section through mouse abdomen Two-level holding stage TRENDS in Biotechnology Figure 1. Schematic representations of ultrasound biomicroscopy (UBM) systems for in utero imaging of mouse embryos UBM-guided Doppler system for imaging and blood velocity analysis. Mice were anesthetized, the lower abdomen and back regions shaved, and a water bath fitted onto the skin. The mouse was laid in the lower level of a two-level stage, and a 100 mm diameter plastic petri dish, modified by punching a 25 mm diameter hole in the center, was pinned over the mouse and filled with water. The 100 mm water bath was used for scanning the UBM imaging transducer and positioning the Doppler transducer for measuring blood velocity. The temperature of the water and mouse (rectal) were maintained at 37 C during Doppler measurements, using heating strips and a feedback temperature controller. UBM-guided injection system for direct manipulation of mouse embryos. Mice were anesthetized, the abdomen shaved, and a 2 cm ventral midline incision made through the skin and peritoneum. A 35 mm diameter, thin rubber membrane was stretched over a 25 mm central hole punched in a 100 mm diameter petri dish. This modified petri dish was pinned over the mouse, gently pulling part of the uterus containing one to two embryos, through a slot cut in the rubber membrane, into the bath of sterile phosphate buffered saline (PBS). A UBM transducer was scanned over the uterus in the PBS, using real-time UBM imaging to guide an injection needle through the uterine wall and into selected embryonic target regions. After injection, the injected embryos were gently pushed back into the abdominal cavity and one to two new embryos were pulled into the PBS for injection. After injecting some or all of the embryos in the litter, the mouse was sutured and allowed to recover in a warming chamber. Parts and reproduced, with permission, from Refs [6] and [23], respectively. myocardial infarction [10] and ventricular function [11]. Applications of conventional phased array ultrasound in the mouse will undoubtedly continue to improve as clinical array transducers and signal processing in these systems use higher frequencies. Although conventional ultrasound provides useful information in the adult mouse, resolution is marginal, and observations of the embryonic and early postnatal heart are virtually impossible; investigations are limited to lowfrequency Doppler measurements of blood velocity and no image confirmation of embryonic orientation [12]. To achieve sufficient resolution to examine the developing mouse heart, MHz UBM has been used for cardiac imaging of mice from early embryonic [3,6,13,14] to neonatal stages [15,16] (Fig. 2a,b). In addition to providing an in vivo approach for analyzing normal heart development, UBM has also been used to detect in utero heart failure in mutant mice that have defects in placental circulation [13], and for analyzing both lethal [15] and nonlethal [16] dilated cardiomyopathy in mutant mice during the first week after birth. UBM imaging has also been extended to include highfrequency (40 50 MHz) continuous wave [17], pulsed wave [18] and color Doppler [19,20], enabling noninvasive blood-velocity measurements to be taken and microcirculatory flow mapping to be carried out (Fig. 2c). UBM image-guided pulsed wave Doppler has been used for precise placement of the Doppler sampling volume for analyzing normal blood velocities in the dorsal aorta and umbilical blood vessels of mouse embryos over a wide range of developmental stages [6,14]. High-frequency color Doppler has also been explored as a means of assessing the microcirculation, particularly in the context of studies on tumor angiogenesis [20,21]. The combination of real-time 2D UBM imaging and Doppler blood-velocity analysis provides a unique, noninvasive way of quantifying structure function relationships in mouse development and disease models in vivo; this will ultimately provide a powerful approach for phenotype analysis of transgenic and mutant mice with defects in cardiovascular development. UBM-guided injection of mouse embryos Developmental biologists have traditionally used numerous approaches to manipulate embryos of lower vertebrate species such as frog, fish and chick directly, transplanting cells and micro-injecting DNA, RNA and viruses under an optical microscope. Direct manipulation of the mouse embryo has been difficult or impossible at most stages of development because the embryos are encased inside the maternal uterus. However, UBM provides a unique method for real-time image-guided injection of cells and other S30

3 Figure 2. Ultrasound biomicroscopy (UBM) analysis of cardiovascular development and microcirculation in the mouse 2D diastolic UBM images of right (RV) and left (LV) ventricles, showing the dilated left ventricle of a postnatal mutant mouse at day 6 (right) compared with normal littermate (left). UBM-guided Doppler analysis of umbilical blood flow in an 11.5 day mouse embryo. UBM image (left) shows heart (H), placenta (P) and umbilical vessels (U), with Doppler sample indicated by cross-hatch marks. The Doppler waveform (right) shows clearly separable umbilical arterial (A) and venous (V) velocities. (c) 2D UBM color Doppler image (left) and 3D segmented image of blood flow (right) in a human melanoma xenografted into nude mouse skin. Blood velocities towards (red) and away (blue) from the transducer range from 0 to 8 mm s 1. The blood flow within this tumor (1.5 mm thick) has a complex distribution of blood motions. Scale bars in (c), 1 mm. Parts and (c) reproduced, with permission, from Refs [15] and [21], respectively. (c) RV P LV U H Velocity (mm s 1 ) A V 0 1 Time (s) 2 TRENDS in Biotechnology agents into a variety of tissues over a wide range of mouse embryonic stages (Fig. 3) for example, enabling in utero transplantation of cells into specific regions of the developing mouse brain [22]. UBM also enables genetic gainof-function studies to be carried out, enabling specific genes to be expressed ectopically using transfected cells [23] and retroviruses [24].This approach has found many applications in mouse developmental biology for example, in the study of neural cell migration and fate in the embryonic brain [25], neural cell-type specification [26], and retrospective cell lineage analysis in the developing limb bud [27] and brain [28].The ability to inject mouse embryos with UBM-guidance has made possible several new investigations not possible in lower vertebrates S31

4 Review A TRENDS Guide to Imaging Technologies Trends in Biotechnology Vol. 20 No. 8 (Suppl.), 2002 Figure 3. UBM-guided injections enable direct manipulation of mouse embryos over a wide range of embryonic stages (a,b) UBM images of an 8.5-day-old mouse embryo, showing uterus (u), neural folds (n) and amniotic membrane. The 50 µm diameter tip of the injection needle is indicated by arrows, before and during injection of retrovirus into the amniotic fluid. (c) UBM-guided injection of cells into the hindlimb (h) of a 10.5-day-old mouse embryo. The forelimb (f) and injection needle tip (arrow) are also shown. (d) UBMguided injection of cells into the forebrain medial ganglionic eminence (m) of a 13.5-day-old mouse embryo. The lateral ventricle (lv) and injection needle tip (arrow) are also shown. Scale bars in (d), 1 mm. Parts (a,b) reproduced, with permission, from [24]. Part (c) reproduced, with permission, from [23]. (c) f u n h a (d) lv m including transplantation and direct gain-of-function studies in a mammalian animal system, with extensive genetic information and many defined mutant strains. By using available transgenic and mutant mice, genetic interactions can be analyzed by performing transplantation or ectopic gene expression studies in mutant mouse embryos, or by transplanting mutant cells into normal mouse embryos. Limitations and future applications of UBM Despite the successes of UBM in providing new in vivo analytical tools for developmental biology, notable obstacles still limit the general applicability of this micro-imaging method. The main advantage of UBM, over other in vivo micro-imaging approaches such as magnetic resonance microscopy, is its real-time capability. However, current prototype and commercial UBM scanners are all based on relatively simple scanning devices that have limited the image-acquisition rates to approximately ten images per second. This is barely adequate even at embryonic to neonatal stages where the normal heart rate varies between three and five beats per second. Although quantitative data have been obtained from cardiac studies, by averaging image-derived parameters over several cardiac cycles in embryonic [13] and neonatal [15,16] mice, and functional data also can be obtained with UBM-guided Doppler [6,14], it would be preferable to obtain UBM images at a higher frame rate. As the normal heart rate of the mouse increases during development, reaching eight to ten beats per second in adult mice, the current frame rate is obviously too slow to enable cardiac dynamics in the adult mouse to be analyzed. UBM would be significantly enhanced if the currently available systems could achieve a level of functionality and ease-of-use similar to that of lower-frequency, clinical ultrasound scanners. Higher frame rates could be achieved either with faster mechanical scanners or by the development of electronically scanned, high-frequency phased array transducers, which represents a significant technical challenge at MHz UBM [29]. High-frequency transducer arrays would also expand the usable depth of field, which is currently limited by the fixed-focus transducers used in UBM systems. In addition to higher image frame rates and electronic scanning, UBM would benefit greatly from the use of small, hand-held high frequency probes, eliminating the need for awkward water-bath or gelstandoff systems currently available.this will be especially important in developing high-throughput UBM approaches for detecting and analyzing mutant phenotypes as part of large-scale mouse mutagenesis screens. UBM in mouse micro-imaging has several strengths. They include its ability to provide noninvasive, in vivo data on a wide range of developmental stages in the mouse S32

5 from embryonic to neonatal and in utero image-guidance for direct injection into mouse embryos. Although the cardiovascular system has been the most-studied organ system to date, in vivo investigations of the development of other organ systems are also possible. For example, UBM has been used to image [30] and measure blood flow in cutaneous tumors in mice [20], and to image the eyes of a mouse model of glaucoma [31]. In addition, it can be used for in vivo studies of disease progression in a wide range of mouse models, including heart and vascular diseases, and tumors in a wide range of superficial and deeper abdominal tissues. UBM therefore provides a versatile and powerful micro-imaging approach for analyzing development and disease in normal and genetically engineered mice. Acknowledgements We would like to thank the past and current members of the Turnbull and Foster laboratories for their participation in the development of the instrumentation and applications described in this review. D.H.T. is supported by grants from the NIH (R01 NS38461), NSF (IBN ) and the McKnight Endowment Fund for Neuroscience. F.S.F. is supported by grants from the National Cancer Institute of Canada and the Canadian Institutes of Health Research. References 1 Sherar, M.D. et al. (1987) Ultrasound backscatter microscopy images the internal structure of living tumour spheroids. Nature 330, Foster, F.S. et al. (2000) Advances in ultrasound biomicroscopy. Ultrasound Med. Biol. 26, Turnbull, D.H. et al. (1995) Ultrasound backscatter microscope analysis of early mouse embryonic brain development. Proc. Natl.Acad. Sci. U. S.A. 92, Sherar, M.D. et al. (1989) A 100 MHz B-scan ultrasound backscatter microscope. Ultrason. Imaging 11, Turnbull, D.H. et al. (1995) A MHz B-scan ultrasound backscatter microscope for skin imaging. Ultrasound Med. Biol. 21, Aristizabal, O. et al. (1998) 40 MHz echocardiography scanner for cardiovascular assessment of mouse embryos. Ultrasound Med. Biol. 24, Foster, F.S. et al. (2000) High frequency ultrasound imaging: from man to mouse. Proceedings of the 2000 IEEE Ultrasonics Symposium, Hoit, B.D. (2001) New approaches to phenotypic analysis in adult mice. J Mol. Cell. Cardiol. 33, Fentzke, R.C. et al. (1998) Dilated cardiomyopathy in transgenic mice expressing a dominant-negative CREB transcription factor in the heart. J. Clin. Invest. 101, Scherrer-Crosbie, M. et al. (1999) Echocardiographic determination of risk area size in a murine model of myocardial ischemia. Am. J. Physiol. 277, H986 H Scherrer-Crosbie, M. et al. (1998) Determination of right ventricular structure and function in normoxic and hypoxic mice: a transesophageal echocardiographic study. Circulation 98, Huang, G.Y. et al. (1998) Alteration in connexin 43 gap junction gene dosage impairs conotruncal heart development. Dev. Biol. 198, Srinivasan, S. et al. (1998) Noninvasive in utero imaging of mouse embryonic heart development using 40 MHz echocardiography. Circulation 98, Phoon, C.K.L. et al. (2000) 40 MHz Doppler characterization of umbilical and dorsal aortic blood flow in the early mouse embryo. Ultrasound Med. Biol. 26, Fatkin, D. et al. (1999) Neonatal cardiomyopathy in mice homozygous for the Arg403Gln mutation in the α cardiac myosin heavy chain gene. J. Clin. Invest. 103, McConnell, B.K. et al. (1999) Dilated cardiomyopathy in homozygous myosin-binding protein-c mutant mice. J. Clin. Invest. 104, Christopher, D.A. et al. (1996) High frequency continuous wave Doppler ultrasound system for the detection of blood flow in the microcirculation. Ultrasound Med. Biol. 22, Christopher, D.A. et al. (1997) High frequency pulsed Doppler ultrasound system for detecting and mapping blood flow in the microcirculation. Ultrasound Med. Biol. 23, Kruse, D. et al. (1998) A swept-scanning mode for estimation of blood velocities in the microvasculature. IEEE Trans. Ultrason. Ferroelec. Freq. Control 45, Goertz, D.E. et al. (2000) High frequency colour flow imaging of the microcirculation. Ultrasound Med. Biol. 26, Foster, F.S. et al. (2000) Ultrasound for the visualization and quantification of tumour microcirculation. Cancer Metastasis Rev. 19, Olsson, M. et al. (1997) Specification of mouse telencephalic and mid-hindbrain progenitors following heterotopic ultrasound-guided embryonic transplantation. Neuron 19, Liu, A. et al. (1998) Alteration of limb and brain patterning in early mouse embryos by ultrasound-guided injection of Shh-expressing cells. Mech. Dev. 75, Gaiano, N. et al. (1999) A method for rapid gain-of-function studies in the mouse embryonic nervous system. Nat. Neurosci. 2, Wichterle, H. et al. (2001) In utero fate mapping reveals distinct migratory pathways and fates of neurons born in the mammalian basal forebrain. Development 128, Gaiano, N. et al. (2000) Radial glial identity is promoted by Notch1 signaling in the murine forebrain. Neuron 26, Kimmel, R. et al. (2000) Two lineage boundaries coordinate vertebrate apical ectodermal ridge formation. Genes Dev. 14, McCarthy, M. et al. (2001) Telencephalic neural progenitors appear to be restricted to regional and glial fates before the onset of neurogenesis. J. Neurosci. 21, Ritter,T.A. et al. (2002) A 30-MHz piezo-composite ultrasound array for medical imaging applications. IEEE Trans. Ultrason. Ferroelec. Freq. Control 49, Turnbull, D.H. et al. (1996) Ultrasound backscatter microscope analysis of mouse melanoma progression. Ultrasound Med. Biol. 22, John, S.W.M. et al. (1998) Essential iris atrophy, pigment dispersion and glaucoma in DBA/2J mice. Invest. Ophthalmol.Vis. Sci. 39, S33