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(Investigative Ophthalmology and Visual Science. 2003;44:2361-2366.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.02-0911
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In Vivo Imaging of Embryonic Development in the Mouse Eye by Ultrasound Biomicroscopy

F. Stuart Foster,1,2 MingYu Zhang,1 Allison S. Duckett,1,2 Viviene Cucevic,1 and Charles J. Pavlin3,4

1From the Sunnybrook and Women’s College Health Sciences Centre, the 2Mouse Imaging Centre at the Hospital for Sick Children, and the 3Departments of Medical Biophysics and 4Ophthalmology, University of Toronto, Toronto, Ontario, Canada.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. New imaging tools now provide an unprecedented opportunity to visualize anatomic and functional development of the mouse eye. In this study, normal embryonic development of the mouse eye was studied by ultrasound biomicroscopy (UBM), with a focus on the formation of the retina, lens, and cornea.

METHODS. The growth of 65 embryonic eyes from timed-pregnant CD-1 mice was examined at various stages of development between embryonic day (E)11.5 and E18.5, using 40-MHz UBM.

RESULTS. The morphogenesis of ocular tissues including the lens, retina, and orbit were revealed from the earliest stages of development. The major axis of the CD-1 lens grows at a rate of 68 µm/d, whereas that of the globe grows at a rate of 122 µm/d, with a concomitant exponential increase in volume.

CONCLUSIONS. UBM allows noninvasive assessment of ocular morphogenesis in vivo and can be used to calculate relative growth rates of ocular structures.

The use of mice as models for the study of human development and disease has a long history extending back to the early 20th century.1 2 However, the availability of drafts of the human and mouse genomes has prompted a rapid acceleration of interest in exploring developmental and disease models based on carefully controlled manipulation of the genetic code. Currently available genetic variants of mice model a wide spectrum of human ocular diseases3 including glaucoma,4 5 retinal and macular degeneration,6 7 8 cataracts,9 10 retinoblastoma,11 and intraocular tumors.12 13 The genetic imprint of disease is often superimposed on the complex molecular expression patterns that accompany normal development. Thus, understanding the regulation and controlling mechanisms of normal development are expected to create knowledge of vital importance for the interpretations of disease states.

Investigation of animal models using the standard tools of optical microscopy and histopathologic analysis are critical benchmarks for the study of development and disease. However, because of the large number of new mutants being produced and the need for rapid, accurate phenotyping, many of the technologies developed for human imaging are now being scaled down for the study of the mouse. This not only permits the opportunity to observe the functional and morphologic results of genetic alteration in vivo but also allows longitudinal studies under carefully controlled conditions. Microimaging technologies, such as fundus photography, fluorescein angiography, and gonioscopy, have been adapted with some difficulty to the mouse.3 Scaled versions of ultrasound imaging,14 15 magnetic resonance (MR) imaging,16 and computed tomographic (CT) imaging17 have also been adapted to image the mouse. Whereas micro-MR and -CT approaches have largely concentrated on imaging of embryonic mouse development after death, ultrasound can be used for in vivo imaging of the mouse embryo. The approaches used for ultrasound imaging are a direct outgrowth of ultrasound biomicroscopy (UBM) currently used in anterior segment imaging in humans.18 UBM is particularly well suited to the study of embryonic development because of its high resolution, comparatively shallow penetration, and rapid imaging times. In this study, UBM techniques were systematically applied to observe ocular development in the mouse embryo. Mice were observed from embryonic day (E)10.5 to E 18.5 to reveal in vivo the normal development of the lens, orbit, retina, and cornea. Quantitative analysis of the growth kinetics of the lens and orbit are presented.


   Methods
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 Abstract
 Methods
Results
 Discussion
 References
 
UBM imaging was performed with a mouse scanner (VS40; VisualSonics, Toronto, Ontario, Canada) operating at a center frequency of 40 MHz with B-scan imaging and Doppler flow measurement capabilities. A schematic of the imaging configuration is given in Figure 1 . The UBM system provided 40 µm axial by 60 µm lateral resolution in an 8 x 8 mm image plane at a 4-Hz frame rate. The gestation period of a mouse is approximately 19 days. Timed pregnancies were determined by vaginal plug observation, with midday time of plug observation counted as E0.5. All animal experimentation was performed under an approved animal care protocol in accordance with the ARVO Statement for the use of Animals in Ophthalmic and Vision Research. Sixty-five embryonic eyes from timed-pregnant CD-1 mice (Charles River, Wilmington, MA) were examined at various stages of development between days E11.5 and E18.5. Mice were anesthetized with isoflurane and imaged on a mouse-imaging stage that provided temperature feedback and heart rate monitoring (THM100; Indus Instruments, Houston, TX). Once the mouse was anesthetized, the abdomen was shaved and further cleaned with a chemical hair remover to minimize ultrasound attenuation. Imaging was performed while maintaining the mouse’s body temperature between 36°C and 38°C with ultrasound gel used as a coupling fluid on the skin. This procedure was performed one to three times on each mouse, with at least 24 hours between imaging sessions. Each mouse imaging study took approximately 2 hours. An example of an image of an E15.5 mouse embryo is shown in Figure 1B . This 8 x 8-mm section shows the placenta (P) and head with a clear view of the embryonic eye (E).



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  		FIGURE 1. (A) UBM setup for use in embryonic mice. Anesthetized mice are scanned at 40 MHz, with a field size of 8 x 8 mm at various stages of gestation. (B) Cross section of the placenta (P) and embryonic head showing the developing eye (E).

 
Mouse embryos tend to have rather random orientations in relation to maternal structure. Selection of embryos to image was based on the visibility of the eyes and the ability to align the transducer axis approximately with the optical axis of the eye. For these reasons it was not practical to attempt repetitive measurements on individual embryos. Images exhibiting the maximum major and minor axes of the oblate spheroidal shape of the lens and orbit were used for measurements made from the outermost lateral boundaries of each echogenic structure studied and captured as records of the analysis.

Ellipsoidal shape assumption is a common approach for estimating volumes from two-dimensional ultrasound images. We assume the ellipsoidal shape of the lens and orbit to be symmetric about the optic axis. Thus, its shape is more correctly referred to as an oblate spheroid because the third dimension of both structures is generally less than the other two dimensions. The volume (V) of an oblate spheroid is calculated by

where a is the maximum dimension of the major axis and c is the maximum dimension of the minor axis. For each day from E11.5 to E18.5, the dimensions of the major and minor axes were tabulated. Based on measurements of between 5 and 10 eyes for each time point from E11.5 to E18.5, the mean volume was calculated using the volume equation and the standard error calculated. Single measurements made at E9.5 and E10.5 are shown in the figures without error bars.


   Results
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 Abstract
 Methods
 Results
Discussion
 References
 
The first evidence of ocular development in the mouse begins at embryonic day 8 at which point the optic placode forms on the inner surfaces of the cephalic neural folds. After the fusion of the neural folds at about E8.5, lateral invaginations of the developing forebrain create optic vesicles from which the eye structures evolve. Visualization of the optic placode and vesicle with UBM was possible at E9.5, as shown in Figure 2A . The optic placode appeared as a thickened indentation of the anterior surface of the forebrain (Fig. 2A , arrow), whereas a day later the optic vesicle was visible as a circumscribed spherical structure with reduced central echogenicity (Fig. 2B) .



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  		FIGURE 2. Early development of the mouse eye. (A) E9.5 image shows a small invagination of the optic placode in the forebrain (arrow) and (B) E10.5 image shows the optic vesicle as an echolucent sphere with a diameter of approximately 250 µm.

 
Neuroectodermal thickening in the lateral wall is destined to become the retina, whereas surface ectodermal thickening gives rise to the lens placode. Between E10 and E11, the lens placode first creates the lens pit and ultimately the lens vesicle. At E11.5 the lens vesicle has developed and appears as a spherical cavity with an echogenic border circumscribed by a narrow hypoechoic rim (Fig. 3A , arrow). At this point there was no evidence of lid, cornea, or retinal development in the UBM image. On E11 and E12, the first evidence of vascular development appears in the primitive optic cup. A groove or choroidal fissure forms, providing access for the hyaloid artery to the optic cup. The hyaloid artery extends anteriorly, branching to form the vascular tunic of the lens. The tissues of the retina develop posteriorly first and subsequently peripherally in a complex series of steps. The latter vascular developments were not visible in the UBM images and did not appear to generate detectable Doppler signals, perhaps because of signal-to-noise problems, which we are investigating for future experiments. Axons from developing retinal ganglion cells extend through the choroidal fissure, forming the optic nerve. Evidence of retinal development could be seen at E13.5, when the posterior aspect of the lens vesicle exhibits a thickening of its echogenic border (Fig. 3C) . At the same time, there is evidence of increasing posterior prominence of the hyperechoic rim. Periocular mesenchymal cells of neural crest origin are thought to give rise to the corneal and conjunctival epithelium. Evidence of corneal development was seen at E14.5 (Fig. 3D) . At that point, the anterior and posterior surfaces of the cornea was reasonably resolved with a thickness of approximately 120 µm. No additional structure in the corneal stroma was visible at this time. At E14.5, the UBM image (Fig. 3D) showed the posterior segment of the lens vesicle splitting into three distinct layers: the developing vitreous cavity (anechoic), immediately posterior to the lens followed by the retina (echoic), and the intraretinal space (anechoic). A fourth layer consisting of the retinal pigment layer is isoechoic and appeared to be less distinguished from the surrounding tissues in the UBM images. The reason for the different backscatter levels observed between various ocular structures remains to be investigated. UBM images of the primary ocular tissues from E14.5 to E18.5 demonstrated progressive morphogenesis. In particular, UBM images of the retina showed continued development and thickening through E18.5 (Figs. 3E 3F 3G 3H) . Delineation of the optic nerve in UBM images can be ameliorated by modifying the angle at which B-mode imaging is performed. However, there was some evidence of this structure from E14.5 to E18.5, as demonstrated in Figures 3D 3E 3F 3G 3H . The development of the lid and iris were difficult to visualize with UBM, as their echogenicity appeared to be similar to that of surrounding tissue.



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  		FIGURE 3. Representative UBM images of embryonic mouse development at E12.5 through E18.5 (A–H, respectively). The growth of normal ocular tissues such as the lens, vitreous, and retina were visualized with resolution on the order of 60 µm. Smallest division, 100 µm.

 
Figure 4 shows a comparison of UBM and histologic sections made at E16.5 and E18.5. A strong concordance between the features just described and the histologic sections was observed. In particular the contrasting scattering properties of the lens,6 vitreous humor,10 retina,5 and posterior retinal space12 were easily identified in the UBM image, whereas less contrasted structures such as the optic nerve,4 cornea, and lid2 were not well visualized. The clear visibility of the lens and the globe of the eye allowed the measurement of growth rates of these prominent structures.



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  		FIGURE 4. Comparison of UBM images (left) at E16.5 and E18.5 with histologic sections from Kaufman (right)19 : 1, conjunctival sac; 2, fused eyelids, hyaloid artery; 4, optic nerve; 5, inner and outer nuclear layers of the retina; 6, lens; 7, anterior chamber; 8, pars iridica retinae; 9, primitive iris; 10, vitreous humor; 11, pigment layer of the retina; 12, intraretinal space; and 13, nerve fiber layer of the optic cup. Illustrations reprinted with permission from Kaufman MH. The Atlas of Mouse Development. London, UK: Academic Press; 1992:404–405. ©Academic Press.

 
Analysis of 65 embryonic eyes in Figure 5 shows a relatively linear growth pattern of the major axis. Least-squares fits of these data have time axis intercepts near 8 days, as expected from previous invasive histologic analyses of mouse eye development.20 The slopes of the fitted growth curves indicate that the major axis of the CD-1 lens has a growth rate of 68 µm per day, whereas the orbit grows at a rate of 122 µm per day. Average major axis dimensions of 0.81 mm in the lens and 1.32 mm in the globe are achieved at E18.5. Plots of lens and globe volume based on average major and minor axis measurements are given in Figure 6 . Mean, range, and standard error of measurements are presented in Tables 1 and 2 .



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  		FIGURE 5. Plots of mouse embryonic lens and globe major axis dimensions. Linear growth was observed during development from E10.5 to E18.5. Error bars indicate SE.

 


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  		FIGURE 6. Plots of mouse embryonic lens and globe volume based on a symmetrical ellipsoidal model. Fits are consistent with a simple exponential growth model. Error bars indicate SE.

 

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  		TABLE 1. Calculated Data from Lens Major Axis Diameter and Volume Measurements

 

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  		TABLE 2. Calculated Values from Globe Major Axis Diameter and Volume Measurements

 

   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
UBM provides a unique means of noninvasively monitoring embryonic ocular development in the mouse. The earliest stages of development are visible by UBM at approximately E8.5, coinciding with the appearance of the optic placode and vesicle. At this point the morphogenesis of the eye is very primitive, and features of the developing tissues are close to the resolution limit of the scanner. Subsequent development of the lens vesicle, retina, cornea, vitreous, and conjunctiva can be observed up to the birth of the mouse. Throughout development until E18.5, anterior segment structures such as the anterior chamber, iris, cornea, and lid appear compressed, revealing little structural detail or differentiation from surrounding tissues. Analysis of growth rates indicate a linear growth pattern of the lens and globe, and an exponential increase in volume. No evidence of flow has yet been observed in the developing prenatal mouse eye. Although this study concentrated on embryonic ocular development, clearly UBM is a useful tool to study the continued development of the mouse eye to adulthood. Extension of these approaches to characterize the mouse eye more fully should provide a valuable means of noninvasively analyzing genetic variants with altered ocular development and disease models.


   Footnotes
 
Supported by the Canadian Institutes for Health Research, National Cancer Institute of Canada with funds from the Terry Fox Foundation.

Submitted for publication September 6, 2002; revised November 29 and December 10, 2002; accepted January 1, 2003.

Disclosure: F.S. Foster, VisualSonics, Inc. (F, I, C, P); M. Zhang, None; A.S. Duckett, VisualSonics, Inc. (C); V. Cucevic, None; C.J. Pavlin, VisualSonics, Inc. (F, I, C, P)

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: F. Stuart Foster, Department of Medical Biophysics and University of Toronto, Sunnybrook and Women’s College Health Sciences Centre, Room S-658, 2075 Bayview Avenue, Toronto, Ontario, Canada M4N 3M5; stuart.foster{at}swchsc.on.ca.


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

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  2. Murray, JA. (1911) Cancerous ancestry and the incidence of cancer in mice Proc R Soc Lond 84,42-48[Free Full Text]
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  4. Anderson, MG, Smith, RS, et al (2002) Mutations in genes encoding melanosomal proteins cause pigmentary glaucoma in DBA/2J mice Nat Genet 30,81-85[CrossRef][Medline][Order article via Infotrieve]
  5. Chang, B, Smith, RS, Hawes, NL, et al (1999) Interacting loci cause severe iris atrophy and glaucoma in DBA/2J mice Nat Genetics 21,405-409[CrossRef][Medline][Order article via Infotrieve]
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  8. Kedzierski, W, Nusinowitz, S, Birch, D, et al (2001) Deficiency of rds/peripherin causes photoreceptor death in mouse models of digenic and dominant retinitis pigmentosa Proc Natl Acad Sci USA 98,7718-7723[Abstract/Free Full Text]
  9. Ai, Y, Zheng, Z, O’Brien, JA, et al (2000) A mouse model of galactose-induced cataracts Hum Mol Genet 9,1821-1827[Abstract/Free Full Text]
  10. Smith, RS, Sundberg, JP, Linder, CC. (1997) Mouse mutations as models for studying cataracts Pathobiology 65,146-154[CrossRef][Medline][Order article via Infotrieve]
  11. Windle, JJ, Albert, DM, O’Brien, JM, et al (1990) Retinoblastoma in transgenic mice Nature 343,665-669[CrossRef][Medline][Order article via Infotrieve]
  12. Syed, NA, Windle, JJ, Darjatmoko, SR. (1998) Transgenic mice with pigmented intraocular tumors: tissue of origin and treatment Invest Ophthalmol Vis Sci 39,2800-2805[Abstract/Free Full Text]
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