February 2003
Volume 44, Issue 2
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Cornea  |   February 2003
Centripetal Movement of Corneal Epithelial Cells in the Normal Adult Mouse
Author Affiliations
  • Takayuki Nagasaki
    From the Department of Ophthalmology, Columbia University, New York, New York.
  • Jin Zhao
    From the Department of Ophthalmology, Columbia University, New York, New York.
Investigative Ophthalmology & Visual Science February 2003, Vol.44, 558-566. doi:10.1167/iovs.02-0705
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      Takayuki Nagasaki, Jin Zhao; Centripetal Movement of Corneal Epithelial Cells in the Normal Adult Mouse. Invest. Ophthalmol. Vis. Sci. 2003;44(2):558-566. doi: 10.1167/iovs.02-0705.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To study the natural movement of corneal epithelial cells in the normal adult mouse with histology and in vivo microscopy.

methods. A transgenic mouse line that was engineered to ubiquitously express green fluorescent protein (GFP) was used to visualize corneal epithelial cells. For histology, epithelial GFP was imaged in a wholemounted cornea en face, and also in frozen cross-sections, under a fluorescence microscope. For in vivo studies, the anesthetized mouse was placed on a custom-made observation platform under a fluorescence microscope. Epithelial fluorescence was digitally recorded two to three times a week, and a rate of cell movement was determined from the time-lapse sequences.

results. The GFP expression in the corneal epithelium was nearly ubiquitous up to about 1 week after birth, and thereafter it gradually became sporadic, resulting in a mosaic pattern of GFP positive cells, with the brightest cells present in the basal and suprabasal layer of the epithelium. Both high- and low GFP-cells formed radial streaks toward the central cornea, frequently displaying vortex patterns at the center. Clusters of several high-GFP cells were tracked in living mice for up to 7 weeks, and an analysis of time-lapse sequences revealed that they moved centripetally at an average rate of 26 μm/d.

conclusions. Corneal epithelium of adult GFP mice exhibits a pattern of GFP expression that is suitable for studying cell movement in the normal cornea. Epithelial cells at the basal or suprabasal layers move centripetally in these mice at an average rate of 26 μm/d.

Corneal epithelium is a dynamic tissue in which cells are constantly renewed and lost, and yet the total mass is kept steady by a mechanism as yet to be elucidated. Continual maintenance of epithelium is essential for its physiological functions, and our understanding of it at a molecular and cellular level would be crucial in coping with various pathologic conditions such as persistent defect and wound healing. 
Cell migration is one of the most fundamental aspects of epithelial homeostasis, and there has been a steady increase in our knowledge in this area. A strong body of evidence now suggests that corneal epithelial cells arise from the stem cells at the limbus. 1 2 3 4 5 6 Once inside the cornea, epithelial cells are thought to move slowly toward the center, as delineated by the X, Y, Z hypothesis of Thoft and Friend. 7 Evidence in support of this concept has been provided by various clinical observations. 8 9 10 11 In addition, a direct observation in humans was reported by Auran et al., 12 who showed, using specular confocal microscopy, that some basal epithelial cells migrate centripetally, 23, 29, and 32 μm over a 24-hour period in three measurements in one eye. However, it remains to be determined whether these short, limited observations can be expanded to show long-term movement of epithelial cells. 
Studies in the animal have also been instructive. Kinoshita et al. 13 presented the first experimental evidence of centripetal movement of cells after lamellar keratoplasty in the rabbit, although their experiments did not address cell movement in the normal cornea directly. Buck 14 was the first to demonstrate the centripetal movement of epithelial cells in the normal cornea. He labeled epithelial cells of the mouse cornea with India ink and determined the rate of movement to be approximately 17 μm/d. However, the observations lasted only 7 days, and the total measured distance was approximately 120 μm, which is less than 10% of the radius of a typical mouse cornea. Thus, it is unclear whether this short-term, short-distance observation can be extrapolated to the general movement of epithelial cells. Also uncertain is whether the labeling with India ink altered the behavior of the cells. 
Thus, despite the evidence suggesting centripetal movement of epithelial cells in the normal cornea, there have been only two studies that presented a direct observation of such movement. 12 14 As such, many basic questions remain unanswered, such as whether the movement is continuous or intermittent, whether it is operative in all areas of the cornea, which layer of epithelium moves at what rate, what the driving force is, and how migration and mitosis are coordinated. 
There has not been much investigation concerning movement of epithelial cells in the normal cornea, presumably because no suitable methodology was available to examine slow movement of cells. Histologic studies are not always appropriate for investigating dynamic events such as cell movement. Accordingly, we sought to establish an animal model in which the migration of epithelial cells could be studied in the uninjured, normal cornea. We report that, by using the GFPU mouse in conjunction with the in vivo microscopy system we developed previously, we were able to observe directly the centripetal movement of corneal epithelial cells for up to 7 weeks in a living mouse. This experimental system should be valuable for further studies on epithelial cell migration in the normal cornea. 
Materials and Methods
Animals
Animal studies adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the institutional animal care and use committee. GFPU mice, which express green fluorescent protein (GFP) ubiquitously under a β-actin promoter, 15 were obtained from the Jackson Laboratory (Bar Harbor, ME), and the line was maintained in our institutional animal facility. Only male mice were used in this study. The sex of newborn mice was determined by opening the abdomen to examine their reproductive organs after death. 
Histology
The eyes of GFPU mice at various ages were isolated after the animal was killed with pentobarbital (100 mg/kg), fixed in formaldehyde, and stained with the nuclear fluorescence dye 4,6-diamidino-2-phenylindole (DAPI), and the corneal size determined. For this, the eyeball was placed under an upright fluorescence microscope (Axioskop2; Carl Zeiss, Oberkochen, Germany) with the cornea facing up. The DAPI image was acquired digitally (Orca, Hamamatsu, Japan) and analyzed on computer (Photoshop; Adobe Systems, San Jose, CA). The corneal boundary was defined as the line across which nuclear density increases sharply, indicating the epithelial cell transition from cornea to limbus. The corneal size was determined by measuring the distance of two perpendicular chords of the cornea and averaging them. It should be noted that this is not surface distance on the cornea but rather the longest linear distance between two margins of the cornea that is naturally situated in the eye. 
Epithelial GFP was digitally recorded with the fluorescence microscope after dissecting the cornea from an eye and making several radial cuts so that it could be mounted flat. 
Some corneas were snap frozen, and 8-μm thick cross-sections were prepared with a cryostat (CM1850; Leica, Wetzlar, Germany). The cryosections were stained with DAPI and digitally recorded with the fluorescence microscope for both DAPI and GFP. 
In Vivo Microscopy
Technical details of in vivo microscopy of living mouse cornea will be reported elsewhere (Maurice D, Zhao J, Nagasaki T, manuscript in preparation). The microscope for the histology, a standard upright fluorescence microscope, as described earlier, was used with a 40× water-immersion (numerical aperture [NA], 0.8), a 10× dry (NA, 0.25), and a 4× dry (NA, 0.1) objective. Mice were anesthetized with isoflurane in oxygen. Anesthesia was maintained by supplying steady flow of anesthetic gas to the nose of the mouse through a nose cone. Before microscopic observation, topical xylazine (20 mg/mL in saline) was given to stimulate proptosis of the eye, 16 so that a wide area of the corneal surface, including the limbus, could be observed without forcefully opening the eyelid. In some mice, when the pupil became dilated, 1% pilocarpine was given topically to promote miosis. This was necessary because strong fluorescence from lens GFP interfered with the observation of corneal fluorescence. 
To reduce motion blur introduced by the mouse’s breathing, its head was held and immobilized with an upper-jaw clamp, which was then magnetically secured to a mouse holder so that the cornea was positioned directly under, and facing toward, the objective lens. When the 40× objective was used, the cornea was lightly touched with a glass coverslip at the bottom of a conical spacer, which was secured on the microscope’s stage, to suppress eye movements in the z direction. The glass applanation also helped flatten the cornea so that a nearly entire field of observation (430 × 340 μm after digitization) was in focus. The mouse was placed on a platform attached to custom-made gimbals that could be rotated freely in all directions, which facilitated rapid scanning of a wide area of the cornea (Maurice D, Zhao J, Nagasaki T, manuscript in preparation). 
Microscopic images were captured with a digital camera (Orca) through a relay zoom lens (0.4× to 2.0×; Carl Zeiss). To minimize phototoxicity to live cells, an illumination source (100-W Hg arc lamp) was operated at 50% or less of its full power. A narrow band-pass excitation filter (480 ± 20 nm; Chroma Technology, Brattleboro, VT) also helped to reduce unnecessary illumination. In addition, a mechanical shutter for the arc light (Uniblitz; Vincent Associates, Rochester, NY) was used to ensure that corneal exposure to light was minimal. The camera and the shutter were synchronously controlled by an image-processing package (Metamorph; Universal Imaging Corp., West Chester, PA), which was also used for postacquisition image processing. 
For routine image acquisition, a few images from different angles were taken with a 4× objective with 0.5× zoom , and 5 to 15 overlapping microscopic fields with a 10× objective with 0.5× zoom. In some animals, 10 to 20 overlapping fields were recorded with a 40× objective with 0.5× zoom to document the details of the corneal surface, which allowed analysis of individual epithelial cells. However, the 40× objective was used only sparingly to minimize the possibility of illumination damage to epithelial cells. A wide-field view was reconstructed from the overlapping images (Photoshop; Adobe). With 10× images, only the central one half to one third of each image was used, because the peripheral zone was out of focus. This assured that a reconstructed image was suitable for measurement of actual corneal surface distance. Under these conditions, image resolution was approximately 3.4, 1.3, and 0.33 μm/pixel for images acquired with a 4×, 10×, and 40× objective, respectively. 
Cell-Tracking and Analysis
The cell movement was analyzed on computer (Photoshop; Adobe) from reconstructed images taken with either a 10× or a 40× objective. A GFP-positive epithelial cell cluster with a unique geometric pattern was chosen for tracking. A composite image of the recorded area was displayed on a computer monitor and either a leading edge or a trailing edge of the chosen cluster was marked and saved as an overlay image. Most of the limbal capillary vessels were strongly GFP positive and served as a fixed reference marker. Two or more marker locations were defined from uniquely shaped capillary branches at an area of limbus nearest to the epithelial cells to be tracked and saved in the same overlay image. The epithelial movement was measured in reference to one of these marker points. When the main marker was not identified, its location was estimated from the other markers. 
Results
Developmental Expression of GFP in the Corneal Epithelium
Expression of GFP in the cornea was predominantly in the epithelium. Contribution from the stroma was negligible when the cornea was observed en face and the epithelium was on the focal plane, both in vivo and ex vivo. Occasionally, endothelial fluorescence was detected as fuzzy background, but its out-of-focus image could be clearly distinguished. Although expression of GFP was ubiquitous in the GFPU mouse, 15 our observation in adult mice revealed that all corneal epithelial cells were not strongly positive with GFP. 
To investigate whether the level of expression was dependent on development, we looked at the epithelial level of GFP as well as the size of the cornea in the GFPU mice at various ages. The nominal size of the cornea doubled in the first 2 weeks after birth, expanding more than 100 μm/d, and reaching the maximum size at 8 to 10 weeks (Fig. 1)
The cornea of a newborn mouse at day 0 appeared almost uniformly positive with GFP (Fig. 2) . In the 7-day-old cornea, however, GFP distribution was no longer uniform, and a mosaic pattern developed that was due to variable levels of expression of GFP. At 2 weeks, when the eyelid opens in these mice, the patchy mosaic pattern was pronounced, with individual patches becoming larger. In the 4-week-old cornea, the mosaic pattern persisted in the central cornea, but there was an emergence of radial patterns of GFP-positive cells that formed stripes at the peripheral cornea. 
In the 8-week-old mouse, in which the corneal size was nearing the maximum, patterns of radial stripes from the periphery toward the center were more obvious, although the stripes did not quite reach the center of the cornea. In this cornea, there appeared to be roughly three levels of GFP expression: high, moderate, and low (hereafter referred to as high-, moderate-, or low-GFP cells). Streaks of moderate-GFP cells were flanked by those of low-GFP cells, and together they defined radial patterns of epithelial expression of GFP. Even though the low-GFP cells appeared dim, these cells clearly contained a detectable level of GFP that was made clear by digital contrast enhancement (data not shown), and no cells appeared to be completely negative with GFP. The high-GFP cells were mostly concentrated in the central part of the cornea, as detailed in the high-magnification view (Fig. 3) . These cells formed clusters of several cells in each and appeared to stand out because of the brightness and clear demarcation. The size of individual high-GFP cells was approximately 10 to 15 μm, suggesting that these cells belonged to the basal or suprabasal layer of the epithelium. The 14-week-old cornea was similar to the 8-week-old cornea (Fig. 2) , in that both contained radial stripe patterns of moderate- and low-GFP cells and also sporadic high-GFP cell clusters. Thus, a general pattern of epithelial expression of GFP in the GFPU mice seems to be nearly established by approximately 8 weeks after birth. 
Also evident was the presence of swirling, or vortex, patterns near the central cornea in the mouse at 4 weeks or older (Fig. 3) , which was frequently made obvious by high-GFP cell clusters concentrated in the central cornea. Detailed patterns of swirling cell distribution generally extended to the very center of the whorl (Figs. 3B 3D 3F 3H) , suggesting the presence of an intricate mechanism. When eight animals (16 eyes) of age 4 to 24 weeks were chosen randomly to score the whorl direction, four eyes showed a clockwise pattern, six eyes counterclockwise, and six eyes contained an ambiguous pattern or none. There was no clear correlation between the two eyes of the same animal. 
Examination of fixed corneas with high-GFP cells revealed that some of these cells in the basal epithelium were going through mitosis (Fig. 4) , indicating that the high-GFP cells were competent for mitosis and that their progeny were likely to maintain a high-GFP phenotype. 
Vertical Location of GFP-Positive Cells
To confirm the vertical location of high-GFP cells within the epithelium, the cornea was fixed and stained with a nuclear dye, DAPI, which helped to define the top and the bottom of the epithelium. The DAPI stain also allowed discrimination of epithelial layers based on the different sizes of the nuclei. The brightest GFP was focused on the basal and suprabasal layer, but not on the superficial layer or anywhere in the upper 17 μm of the 25-μm epithelium (Fig. 5) , consistent with a conclusion that the high-GFP cells were located in the basal or suprabasal layer. The demarcation of suprabasal cells is clearer than that of the basal cells, probably because cytoplasm of the former is much thinner and therefore contribution of out-of-focus fluorescence is less. 
To examine a cross-sectional view of these cells, a cryosection was prepared from a piece of cornea whose en face profile had been recorded previously. This allowed a direct comparison between an en face view and a cross-sectional view of the same cells. The results show that the cells that appear bright in an en face view were primarily located at the basal or suprabasal layers (Fig. 6) . There were some superficial cells with bright GFP in a cross-section, but the fluorescence of these cells in an en face view was likely to be overwhelmed by the basal and/or suprabasal layer cells that had much thicker cytoplasm than the superficial cells. Thus, the combined results of Figures 5 and 6 strongly suggest that the high-GFP cells that we identified in an en face view, as with in vivo microscopy (described in the next section), were located in the basal and suprabasal layers of the epithelium. It is possible that bright cells existed at the superficial layer temporarily, but they would have been lost by desquamation rapidly within days. 
In Vivo Observation of GFP-Positive Epithelial Cells
Because high-GFP cells were sporadic and they formed a unique geometric shape, we were able to relocate them at different times without difficulty, and therefore track their movement in the living mouse. To generate time-lapse sequences, epithelial GFP was imaged at an interval of two to three times per week. One such recording is shown in Figure 7 , in which an elongated cluster of high-GFP cells can be seen migrating from the limbus nearly straight to the central cornea. The overall shape of the cell cluster seemed to change only slightly as it moved, and no substantial alteration was noted. It was not possible to follow up further movement into the central cornea, because strong background fluorescence from lens GFP above the pupil interfered with imaging of corneal GFP. 
Another time-lapse sequence is shown in Figure 8 , in which centripetal movement of three high-GFP cell clusters is obvious (Figs. 8B 8E 8H 8K ; white box). High-power images of these cells (Figs. 8C 8F 8I 8L) show that the relative positions of three clusters did not change, but that the individual cells were not stationary within the clusters. Such local mobility was not studied further, because much shorter intervals of observation were necessary. At the end of this recording, the cornea was fixed and examined for the vertical location of high-GFP cells, similar to Figure 5 , and they were found to be in the basal to suprabasal layer (Fig. 9) . The size of these high-GFP cells, less than 20 μm, is consistent with their being located in the basal and suprabasal epithelium. 
Clusters of moderate-GFP cells (e.g., Fig. 8 , arrows) also exhibited centripetal movement, but the rate of their movement appeared to be slower. However, tracking of these cell clusters was not attempted, because they were generally large and undefined in shape, and therefore it was difficult to identify a position that can be accurately relocated for tracking. 
To evaluate the cell movement quantitatively, the location of selected high-GFP cell clusters was determined at various time points in reference to a fixed marker at the limbus in seven recordings from four corneas (Fig. 10) . The plots show that the rate of cell movement was fairly constant throughout the cornea, from the periphery to the central zone. Rates of movement were determined from these plots (Table 1) , and the average rate of centripetal movement was found to be approximately 26 μm/d. The steady rates of movement over several weeks suggest that any illumination-induced phototoxicity was minimal. 
In all the seven recordings that were analyzed, movement of high-GFP cells was always toward the center, although the precise level of deviation from a straight line was not determined. It was also noted that high-GFP cell clusters never emerged suddenly in the middle of the cornea during the recordings. Either they were already present in the middle of the cornea at the beginning of the recording, or they appeared at the peripheral margin of the cornea from the limbus, as was the case in Figure 7
Discussion
GFP Expression in the Corneal Epithelium
Use of the GFPU mouse in conjunction with in vivo microscopy allowed us to investigate the movement of epithelial cells in the normal cornea. In the adult GFPU mouse, only some of the epithelial cells were highly positive with GFP, which was ideal for cell tracking, because these cells could be easily relocated at different times. The reason for the variable GFP expression is not clear, but similar observations were reported recently by Collinson et al. 17 who examined lac-Z–expressing epithelial cells in chimeric mice. 
Expression of GFP in the young mouse, 4 weeks or younger, was not stable, but in older animals, expression levels of GFP in limbal stem cells seemed to be generally maintained in daughter cells, as evidenced by the presence of radial stripes that emerged from the limbus (Fig. 2) . Furthermore, our results in Figure 7 indicate that a cluster of high-GFP cells remained high in GFP over 6 weeks, which would be long enough for a few generations of these cells. These observations, though indirect, suggest that the limbal stem cells express different levels of GFP and that this trait is inherited by the daughter cells and further by their progeny, at least in the 7- to 15-week-old adult mice that we used for measurement of centripetal movement of cells. 
Centripetal Movement of High-GFP Cells
Radial patterns of epithelial GFP in histologic specimens (Fig. 2) first gave a hint of centripetal movement of cells, similar to many clinical observations. 9 11 Similar patterns were observed by Collinson et al. 17 in lac-Z chimeric mice, which exhibited dramatic and unmistakable stripes. The radial patterns were not obvious, however, during the first 2 weeks after birth, when the cornea grew at a rate of more than 100 μm/d under the closed eyelid (Fig. 1) . It seems that centripetal movement of cells in a direction opposite that of corneal growth was completely negated by the rapid growth of the cornea. When the radial patterns became well established in the adult mouse, aged approximately 8 weeks and more, high-GFP cell clusters were generally more concentrated in the central portion of the cornea, which is consistent with their centripetal movement and convergence in the center. 
In vivo microscopy provided a more direct and convincing evidence of centripetal movement of cells (Figs. 7 8 10) . The rate of movement was approximately 26 μm/d (Table 1) , which is comparable to the previous findings in the mouse 14 and the human 12 (see the introduction). 
Vortex Pattern of GFP
Some of the corneas exhibited vortex patterns in the central cornea, which were also present in the lac-Z chimeric mice, 17 but how they came to exist is not clear. Developmentally, appearance of central vortex patterns coincided with that of radial stripes at the peripheral cornea, approximately 4 weeks after birth, suggesting that centripetal movement of cells may be involved in the formation of the vortex. Thus, it is plausible that the vortex is a natural consequence of many cells converging in a small central area from all directions, as originally suggested by Bron. 9 If this is true, a constant migration cue, or a driving force, in the centripetal direction should be assumed, possibly a population pressure generated at a peripheral zone of the cornea. However, there were some corneas in which no vortex could be identified, despite the unequivocal presence of radial stripes (data not shown), suggesting that formation of the vortex may require more than a simple pressure from the periphery. 
In humans, the whorl pattern was almost always found to be clockwise, and electromagnetic fields of the eye have been suggested as the cause. 18 However, the whorls in the GFPU mouse cornea were both clockwise and counterclockwise, similar to those in the Lac-Z chimeric mice, 17 and therefore the electromagnetic theory does not seem applicable to these mice. 
Mitosis and the Centripetal Movement of Cells
We observed movement of high-GFP cells from limbus to central cornea over 7 weeks (Figs. 7 10) . If the turnover time of epithelial cells was 2 weeks, 19 there would have been at least three generations of high-GFP cells during this period. A shorter generation time 20 21 would have required more frequent mitoses within the high-GFP cell cluster. Therefore, the observed high-GFP cells during the 7-week period were not the same cells, but they collectively represented the progeny of high-GFP basal cells that were present at the beginning of the recording. The occurrence of multiple mitoses within the high-GFP cell cluster indicates that the observed centripetal movement, averaging approximately 26 μm/d (Table 1) , was a combined result of cell division and migration. 
The size of high-GFP cell clusters, thus the number of these cells, appeared unchanged throughout their movement for a long distance. This suggests that every mitosis resulted in, on the average, one surviving high-GFP cell, which is in agreement with an earlier suggestion. 21 In this context, it is interesting to note that postmitotic cells were almost always found as a pair of daughter cells in proximity to each other in both basal and wing cell layers. 22 If this were true of high-GFP cells, it would suggest that one pair of high-GFP daughter cells survives after every two mitoses, instead of one daughter cell surviving from a single mitosis. The other pair of high-GFP daughter cells were probably lost from the surface rapidly, so as to maintain the size of the high-GFP cell cluster. 
Alternatively, a possibility exists that all the high-GFP cells that we tracked (Table 1) were replication incompetent and that the observed high-GFP cells were in fact the same epithelial cells throughout the recordings. We think this to be unlikely, however, because our results demonstrated that high-GFP cells could go through mitosis (Fig. 4)
Driving Force
The driving force of the centripetal movement of cells is not known, although several proposals have been advanced. There are four viable published hypotheses, which are not mutually exclusive: (1) preferential desquamation of central corneal epithelium, drawing peripheral cells toward the central cornea 11 23 ; (2) population pressure from limbus and peripheral cornea due to proliferation and immigration of cells 4 9 24 ; (3) a gradient of chemical signals emanating from limbal capillary vessels 14 ; and (4) stimulation by epithelial sympathetic nerves. 25  
Although our observations did not provide any direct evidence, they suggest that population pressure plays an important role. We observed fine vortex patterns at the center of the normal cornea (Fig. 3) , which we believe is best explained by the population pressure theory discussed earlier. We also observed that the general shape of high-GFP cell clusters and their positions relative to each other were maintained while they moved centripetally (Figs. 7 8) . This may be an indication that there was a tightly regulated external force, such as that provided by population pressure, that positively guided the cells for directional movement. A source of population pressure can be cell division and/or cell migration. However, some have claimed a higher mitotic rate in the peripheral cornea compared with the central cornea, 17 20 26 27 28 whereas others have reported similar rates between them, 23 29 30 31 32 and consequently the contribution of cell division remains unclear. If the latter possibility is true, population pressure must be provided by continual migration of cells from the limbus into the cornea. 
Regardless the role of population pressure, the other three hypotheses remain a possibility at this time. In particular, we are attracted by a stromal molecular gradient originating from the limbal capillaries 33 as a possible contributor to the centripetal movement of cells. 14  
Homeostasis of Normal Corneal Epithelium
Although dynamics of cell renewal in the stratified epithelium is complex, a relatively simple picture emerges from the results of earlier investigations as well as the present study. As an extension of the X, Y, Z theory, 7 we envisage five major components in the epithelial homeostasis of normal cornea: (1) generation of transient amplifying cells from the stem cells and their entrance into the cornea 1 2 3 4 5 6 ; (2) cell divisions at the basal epithelium 19 26 29 ; (3) vertical movement of basal cells and their daughter cells toward the surface, which is associated with terminal differentiation 4 20 21 22 34 ; (4) horizontal movement of cells toward the central cornea 7 12 13 14 (present study); and (5) loss of surface cells by exfoliation. 35  
Basal cell division and vertical movement of cells have been well characterized, but the other three are relatively poorly understood. In this study, the centripetal movement of epithelial cells occurred at a steady rate of approximately 26 μm/d (Fig. 10 , Table 1 ), for almost an entire distance of the corneal radius in one case (Fig. 7) . The steady centripetal movement would cause overcrowding in the central cornea unless cells were removed as rapidly as they accumulate. 7 In fact, experimental evidence suggests that surface cells are lost preferentially in the central cornea. 35 36 Although the role of central desquamation in centripetal movement of cells is not certain, it is clearly an essential process for epithelial homeostasis. 
The steady rate of centripetal movement of cells also suggests that the limbal stem cell progeny enters the cornea at a similar rate, although this has not been observed directly in the normal eye. Consequently, little is known about the nature of this immigration process, such as rate, frequency, and location. We believe it is now possible to investigate these parameters with the technique used in this study by continuously tracking the movement of high-GFP cells in the limbus. 
The question now is how these distinct events are coordinated or how each of the five components influences the outcome of others, for the common goal of maintaining the epithelial mass. A mathematical model 24 may be a useful approach if the parameters for these components could be determined experimentally. Some pathologic conditions, such as persistent defects and wound healing may be better understood by examining these five components separately. 
In summary, the GFPU mouse offers a unique opportunity to study movement of epithelial cells in the normal cornea. Further microscopic analyses of these corneas, combined with various biochemical markers of epithelial differentiation, 4 37 should be useful in understanding epithelial homeostasis. 
 
Figure 1.
 
Increase in corneal size in the GFPU mouse. The corneal size is represented by a nominal diameter of the cornea, which is defined as a chord, or the longest linear distance between two margins of a cornea that is naturally situated in the eye. The surface distance is longer than the chord because of the corneal curvature.
Figure 1.
 
Increase in corneal size in the GFPU mouse. The corneal size is represented by a nominal diameter of the cornea, which is defined as a chord, or the longest linear distance between two margins of a cornea that is naturally situated in the eye. The surface distance is longer than the chord because of the corneal curvature.
Figure 2.
 
Developmental expression of GFP in the corneal epithelium. Fixed corneas were slit, flattened, and observed under a fluorescence microscope. White: GFP-positive areas. Shown are representatives of at least two eyes at each age. Central areas of the corneas at 4-, 8-, 14-, and 24 weeks are shown magnified in Figure 3 .
Figure 2.
 
Developmental expression of GFP in the corneal epithelium. Fixed corneas were slit, flattened, and observed under a fluorescence microscope. White: GFP-positive areas. Shown are representatives of at least two eyes at each age. Central areas of the corneas at 4-, 8-, 14-, and 24 weeks are shown magnified in Figure 3 .
Figure 3.
 
Vortex patterns in the central cornea of GFP mice. Images are magnified views of the (A, B) 4-, (C, D) 8-, (E, F) 14-, and (G, H) 24- week corneas that are shown in Figure 2 . Central areas of images in (A, C, E, G) were magnified four times and are shown in (B, D, F, H), respectively.
Figure 3.
 
Vortex patterns in the central cornea of GFP mice. Images are magnified views of the (A, B) 4-, (C, D) 8-, (E, F) 14-, and (G, H) 24- week corneas that are shown in Figure 2 . Central areas of images in (A, C, E, G) were magnified four times and are shown in (B, D, F, H), respectively.
Figure 4.
 
High-GFP cells that were in mitosis. The corneal epithelium of an 8.3-week-old mouse was stained with DAPI to reveal the mitotic cells. The DAPI (A, C) and GFP (B, D) profile of the same cells are shown. Arrows: mitotic cells in metaphase (A, B) and telophase (C, D). All cells were located in the basal epithelium.
Figure 4.
 
High-GFP cells that were in mitosis. The corneal epithelium of an 8.3-week-old mouse was stained with DAPI to reveal the mitotic cells. The DAPI (A, C) and GFP (B, D) profile of the same cells are shown. Arrows: mitotic cells in metaphase (A, B) and telophase (C, D). All cells were located in the basal epithelium.
Figure 5.
 
Expression of GFP in different layers of the epithelium. Cornea was fixed and stained with DAPI. Double-fluorescence images of the same cells are shown for DAPI (A, C, E) and GFP (B, D, F). The microscope was focused on the top layer (A, B), the suprabasal layer 17 μm from the top (C, D), and the basal layer 25 μm from the top (E, F). Cells with the brightest GFP were located on a plane of the suprabasal and basal epithelium.
Figure 5.
 
Expression of GFP in different layers of the epithelium. Cornea was fixed and stained with DAPI. Double-fluorescence images of the same cells are shown for DAPI (A, C, E) and GFP (B, D, F). The microscope was focused on the top layer (A, B), the suprabasal layer 17 μm from the top (C, D), and the basal layer 25 μm from the top (E, F). Cells with the brightest GFP were located on a plane of the suprabasal and basal epithelium.
Figure 6.
 
A comparison of an en face view (A, B, C) and a cross-sectional view (D) of the same cells with high GFP. A corneal piece from a 16-week-old mouse was mounted flat on a glass slide and photographed under a fluorescence microscope after focused on the top (A), middle (B), and bottom (C) layers of the epithelium. This piece was frozen in embedding compound and serial cryosections of 8-μm thickness were prepared. (D) A cryosection that corresponds to the dotted lines in (A), (B), and (C). Letters at right: corresponding positions of each panel.
Figure 6.
 
A comparison of an en face view (A, B, C) and a cross-sectional view (D) of the same cells with high GFP. A corneal piece from a 16-week-old mouse was mounted flat on a glass slide and photographed under a fluorescence microscope after focused on the top (A), middle (B), and bottom (C) layers of the epithelium. This piece was frozen in embedding compound and serial cryosections of 8-μm thickness were prepared. (D) A cryosection that corresponds to the dotted lines in (A), (B), and (C). Letters at right: corresponding positions of each panel.
Figure 7.
 
A time-lapse sequence of epithelial cell movement at low power (sequence 4-1 in Fig. 10 , Table 1 ). Epithelial GFP fluorescence was recorded with a 4× objective in a living mouse at the indicated times, which represent the age of the mouse. This cornea was tracked for 7 weeks, and images of the last 6 weeks are shown. Arrows: the tip of the bright cluster that was tracked for movement. Traveled distances (Fig. 10 , Table 1 ) were measured in images obtained with a 10× objective (not shown), using limbal capillary markers.
Figure 7.
 
A time-lapse sequence of epithelial cell movement at low power (sequence 4-1 in Fig. 10 , Table 1 ). Epithelial GFP fluorescence was recorded with a 4× objective in a living mouse at the indicated times, which represent the age of the mouse. This cornea was tracked for 7 weeks, and images of the last 6 weeks are shown. Arrows: the tip of the bright cluster that was tracked for movement. Traveled distances (Fig. 10 , Table 1 ) were measured in images obtained with a 10× objective (not shown), using limbal capillary markers.
Figure 8.
 
A time-lapse sequence of epithelial cell movement (sequence 2-1 in Fig. 10 , Table 1 ). Epithelial GFP was recorded in a living mouse, 10 weeks old at the beginning of the recording, over 13 days. Shown are images acquired on days 0 (A, B, C), 4 (D, E, F), 8 (G, H, I), and 13 (J, K, L). At each time point, a low-power image (A, D, G, J), a medium-power image including the limbus (B, E, H, K), and a high-power image of three high-GFP cell clusters (C, F, I, L) are presented. White box: area of the corresponding higher-power images. For medium- and high-power images, overlapping fields were recorded and patched together. The left margin of low- and medium-power images is the limbus, and the central cornea is toward the right. Two white dots at left of the medium power images (B, E, H, K) denote the fixed-position markers as determined by the unique shape of limbal capillary vessels. These vessels appear clearly on a computer monitor after several-fold magnification. (B, E, H, K, arrows) Cells of moderate-GFP cell clusters. The area of the white box in (K) is shown in Figure 9 after fixation of the cornea.
Figure 8.
 
A time-lapse sequence of epithelial cell movement (sequence 2-1 in Fig. 10 , Table 1 ). Epithelial GFP was recorded in a living mouse, 10 weeks old at the beginning of the recording, over 13 days. Shown are images acquired on days 0 (A, B, C), 4 (D, E, F), 8 (G, H, I), and 13 (J, K, L). At each time point, a low-power image (A, D, G, J), a medium-power image including the limbus (B, E, H, K), and a high-power image of three high-GFP cell clusters (C, F, I, L) are presented. White box: area of the corresponding higher-power images. For medium- and high-power images, overlapping fields were recorded and patched together. The left margin of low- and medium-power images is the limbus, and the central cornea is toward the right. Two white dots at left of the medium power images (B, E, H, K) denote the fixed-position markers as determined by the unique shape of limbal capillary vessels. These vessels appear clearly on a computer monitor after several-fold magnification. (B, E, H, K, arrows) Cells of moderate-GFP cell clusters. The area of the white box in (K) is shown in Figure 9 after fixation of the cornea.
Figure 9.
 
Epithelial GFP after fixation of the cornea shown in Figure 8K (white box). The fluorescence microscope was focused on three different levels of epithelium: the superficial (A), suprabasal (B), and basal cell (C) layers, similar to Figure 5 . The focal plane of the brightest cells corresponds to the basal or suprabasal layer of the epithelium.
Figure 9.
 
Epithelial GFP after fixation of the cornea shown in Figure 8K (white box). The fluorescence microscope was focused on three different levels of epithelium: the superficial (A), suprabasal (B), and basal cell (C) layers, similar to Figure 5 . The focal plane of the brightest cells corresponds to the basal or suprabasal layer of the epithelium.
Figure 10.
 
Tracking of GFP-positive epithelial cell clusters in four living mice (seven time-lapse recordings). The labeling, which corresponds to that in Table 1 is, for example, 1-2 denoting mouse 1, sequence 2, and so on. Shown are distances from location markers at the limbus, identified by capillary branches, which were sometimes beyond the margin of the cornea. Also, note that the measurements are of a corneal surface distance, calculated from microscopic images obtained with a 10× objective and do not correspond to the size of the cornea shown in Figure 1 .
Figure 10.
 
Tracking of GFP-positive epithelial cell clusters in four living mice (seven time-lapse recordings). The labeling, which corresponds to that in Table 1 is, for example, 1-2 denoting mouse 1, sequence 2, and so on. Shown are distances from location markers at the limbus, identified by capillary branches, which were sometimes beyond the margin of the cornea. Also, note that the measurements are of a corneal surface distance, calculated from microscopic images obtained with a 10× objective and do not correspond to the size of the cornea shown in Figure 1 .
Table 1.
 
Rate of Centripetal Movement of Corneal Epithelial Cells
Table 1.
 
Rate of Centripetal Movement of Corneal Epithelial Cells
Sequence ID Age (wk) Duration (d) Rate by Total Distance (μm/d) Rate by Regression (μm/d)
1-1 10 18 21.3 22.2
1-2 11 19 31.9 32.1
2-1* 10 13 20.8 22.0
2-2 10 13 21.8 22.3
3-1 7 9 25.6 26.5
4-1, † 7 49 28.0 26.7
4-2, ‡ 11 24 30.4 32.8
Average 25.7 26.4
Authors thank David Maurice for his eternal wisdom and support. 
Schermer, A, Galvin, S, Sun, TT. (1986) Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells J Cell Biol 103,49-62 [CrossRef] [PubMed]
Cotsarelis, G, Cheng, SZ, Dong, G, Sun, TT, Lavker, RM. (1989) Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells Cell 57,201-209 [CrossRef] [PubMed]
Tseng, SC. (1989) Concept and application of limbal stem cells Eye 3,141-157 [CrossRef] [PubMed]
Wolosin, JM, Xiong, X, Schutte, M, Stegman, Z, Tieng, A. (2000) Stem cells and differentiation stages in the limbo-corneal epithelium Prog Retinal Eye Res 19,223-255 [CrossRef]
Dua, HS, Azuara-Blanco, A. (2000) Limbal stem cells of the corneal epithelium Surv Ophthalmol 44,415-425 [CrossRef] [PubMed]
Daniels, JT, Dart, JK, Tuft, SJ, Khaw, PT. (2001) Corneal stem cells in review Wound Repair Regen 9,483-494 [CrossRef] [PubMed]
Thoft, RA, Friend, J. (1983) The X, Y, Z hypothesis of corneal epithelial maintenance Invest Ophthalmol Vis Sci 24,1442-1443 [PubMed]
Davanger, M, Evensen, A. (1971) Role of the pericorneal papillary structure in renewal of corneal epithelium Nature 229,560-561 [CrossRef] [PubMed]
Bron, AJ. (1973) Vortex patterns of the corneal epithelium Trans Ophthalmol Soc UK 93,455-472 [PubMed]
Kaye, DB. (1980) Epithelial response in penetrating keratoplasty Am J Ophthalmol 89,381-387 [CrossRef] [PubMed]
Lemp, MA, Mathers, WD. (1989) Corneal epithelial cell movement in humans Eye 3,438-445 [CrossRef] [PubMed]
Auran, JD, Koester, CJ, Kleiman, NJ, et al (1995) Scanning slit confocal microscopic observation of cell morphology and movement within the normal human anterior cornea Ophthalmology 102,33-41 [CrossRef] [PubMed]
Kinoshita, S, Friend, J, Thoft, RA. (1981) Sex chromatin of donor corneal epithelium in rabbits Invest Ophthalmol Vis Sci 21,434-441 [PubMed]
Buck, RC. (1985) Measurement of centripetal migration of normal corneal epithelial cells in the mouse Invest Ophthalmol Vis Sci 26,1296-1299 [PubMed]
Hadjantonakis, AK, Gertsenstein, M, Ikawa, M, Okabe, M, Nagy, A. (1998) Generating green fluorescent mice by germline transmission of green fluorescent ES cells Mech. Dev 76,79-90 [CrossRef] [PubMed]
Calderone, L, Grimes, P, Shalev, M. (1986) Acute reversible cataract induced by xylazine and by ketamine-xylazine anesthesia in rats and mice Exp Eye Res 42,331-337 [CrossRef] [PubMed]
Collinson, JM, Morris, L, Reid, AI, et al (2002) Clonal analysis of patterns of growth, stem cell activity, and cell movement during the development and maintenance of the murine corneal epithelium Dev Dyn 224,432-440 [CrossRef] [PubMed]
Dua, HS, Watson, NJ, Mathur, RM, Forrester, JV. (1993) Corneal epithelial cell migration in humans: “hurricane and blizzard keratopathy” Eye 7,53-58 [CrossRef] [PubMed]
Haskjold, E, Refsum, SB, Bjerknes, R. (1989) Circadian variation in the mitotic rate of the rat corneal epithelium: cell divisions and migration are analyzed by a mathematical model Virchows Arch B Cell Pathol 58,123-127 [CrossRef]
Hanna, C, O’Brien, JE. (1960) Cell production and migration in the epithelial layer of the cornea Arch Ophthalmol 64,536-539 [CrossRef] [PubMed]
Elgjo, K. (1969) Cell renewal of the normal mouse cornea Acta Pathol Microbiol Scand 76,25-30 [PubMed]
Beebe, DC, Masters, BR. (1996) Cell lineage and the differentiation of corneal epithelial cells Invest Ophthalmol Vis Sci 37,1815-1825 [PubMed]
Lavker, RM, Dong, G, Cheng, SZ, Kudoh, K, Cotsarelis, G, Sun, TT. (1991) Relative proliferative rates of limbal and corneal epithelia: implications of corneal epithelial migration, circadian rhythm, and suprabasally located DNA-synthesizing keratinocytes Invest Ophthalmol Vis Sci 32,1864-1875 [PubMed]
Sharma, A, Coles, WH. (1989) Kinetics of corneal epithelial maintenance and graft loss: a population balance model Invest Ophthalmol Vis Sci 30,1962-1971 [PubMed]
Jones, MA, Marfurt, CF. (1996) Sympathetic stimulation of corneal epithelial proliferation in wounded and nonwounded rat eyes Invest Ophthalmol Vis Sci 37,2535-2547 [PubMed]
Buschke, W, Friedenwald, JS, Fleischmann, W. (1943) Studies on the mitotic activity of the corneal epithelium: methods—the effects of colchicine, ether, cocaine, and ephedrin Bull Johns Hopkins Hosp 73,143-168
Cenedella, RJ, Fleschner, CR. (1990) Kinetics of corneal epithelium turnover in vivo: studies of lovastatin Invest Ophthalmol Vis Sci 31,1957-1962 [PubMed]
Ladage, PM, Yamamoto, K, Ren, DH, et al (2001) Proliferation rate of rabbit corneal epithelium during overnight rigid contact lens wear Invest Ophthalmol Vis Sci 42,2804-2812 [PubMed]
Kaufmann, B, Gay, H, Hollaender, A. (1944) Distribution of mitoses in the corneal epithelium of the rabbit and the rat Anat Rec 90,161-178 [CrossRef]
Fogle, JA, Yoza, BK, Neufeld, AH. (1980) Diurnal rhythm of mitosis in rabbit corneal epithelium Graefes Arch Klin Exp Ophthalmol 213,143-148 [CrossRef]
Haskjold, E, Refsum, SB, Bjerknes, R. (1988) Cell renewal of the rat corneal epithelium: a method to compare corresponding corneal areas from individual animals Acta Ophthalmol 66,533-537
Szerenyi, K, Wang, X, Gabrielian, K, LaBree, L, McDonnell, PJ. (1994) Immunochemistry with 5-bromo-2-deoxyuridine for visualization of mitotic cells in the corneal epithelium Cornea 13,487-492 [PubMed]
Maurice, DM, Watson, PG. (1965) The distribution and movement of serum albumin in the cornea Exp Eye Res 4,355-363 [CrossRef] [PubMed]
Haskjold, E, Bjerknes, R, Bjerknes, E. (1989) Migration of cells in the rat corneal epithelium Acta Ophthalmol 67,91-96
Ren, H, Wilson, G. (1996) Apoptosis in the corneal epithelium Invest Ophthalmol Vis Sci 37,1017-1025 [PubMed]
Mathers, WD, Lemp, MA. (1992) Morphology and movement of corneal surface cells in humans Curr Eye Res 11,517-523 [CrossRef] [PubMed]
Kinoshita, S, Adachi, W, Sotozono, C, et al (2001) Characteristics of the human ocular surface epithelium Prog Retinal Eye Res 20,639-673 [CrossRef]
Figure 1.
 
Increase in corneal size in the GFPU mouse. The corneal size is represented by a nominal diameter of the cornea, which is defined as a chord, or the longest linear distance between two margins of a cornea that is naturally situated in the eye. The surface distance is longer than the chord because of the corneal curvature.
Figure 1.
 
Increase in corneal size in the GFPU mouse. The corneal size is represented by a nominal diameter of the cornea, which is defined as a chord, or the longest linear distance between two margins of a cornea that is naturally situated in the eye. The surface distance is longer than the chord because of the corneal curvature.
Figure 2.
 
Developmental expression of GFP in the corneal epithelium. Fixed corneas were slit, flattened, and observed under a fluorescence microscope. White: GFP-positive areas. Shown are representatives of at least two eyes at each age. Central areas of the corneas at 4-, 8-, 14-, and 24 weeks are shown magnified in Figure 3 .
Figure 2.
 
Developmental expression of GFP in the corneal epithelium. Fixed corneas were slit, flattened, and observed under a fluorescence microscope. White: GFP-positive areas. Shown are representatives of at least two eyes at each age. Central areas of the corneas at 4-, 8-, 14-, and 24 weeks are shown magnified in Figure 3 .
Figure 3.
 
Vortex patterns in the central cornea of GFP mice. Images are magnified views of the (A, B) 4-, (C, D) 8-, (E, F) 14-, and (G, H) 24- week corneas that are shown in Figure 2 . Central areas of images in (A, C, E, G) were magnified four times and are shown in (B, D, F, H), respectively.
Figure 3.
 
Vortex patterns in the central cornea of GFP mice. Images are magnified views of the (A, B) 4-, (C, D) 8-, (E, F) 14-, and (G, H) 24- week corneas that are shown in Figure 2 . Central areas of images in (A, C, E, G) were magnified four times and are shown in (B, D, F, H), respectively.
Figure 4.
 
High-GFP cells that were in mitosis. The corneal epithelium of an 8.3-week-old mouse was stained with DAPI to reveal the mitotic cells. The DAPI (A, C) and GFP (B, D) profile of the same cells are shown. Arrows: mitotic cells in metaphase (A, B) and telophase (C, D). All cells were located in the basal epithelium.
Figure 4.
 
High-GFP cells that were in mitosis. The corneal epithelium of an 8.3-week-old mouse was stained with DAPI to reveal the mitotic cells. The DAPI (A, C) and GFP (B, D) profile of the same cells are shown. Arrows: mitotic cells in metaphase (A, B) and telophase (C, D). All cells were located in the basal epithelium.
Figure 5.
 
Expression of GFP in different layers of the epithelium. Cornea was fixed and stained with DAPI. Double-fluorescence images of the same cells are shown for DAPI (A, C, E) and GFP (B, D, F). The microscope was focused on the top layer (A, B), the suprabasal layer 17 μm from the top (C, D), and the basal layer 25 μm from the top (E, F). Cells with the brightest GFP were located on a plane of the suprabasal and basal epithelium.
Figure 5.
 
Expression of GFP in different layers of the epithelium. Cornea was fixed and stained with DAPI. Double-fluorescence images of the same cells are shown for DAPI (A, C, E) and GFP (B, D, F). The microscope was focused on the top layer (A, B), the suprabasal layer 17 μm from the top (C, D), and the basal layer 25 μm from the top (E, F). Cells with the brightest GFP were located on a plane of the suprabasal and basal epithelium.
Figure 6.
 
A comparison of an en face view (A, B, C) and a cross-sectional view (D) of the same cells with high GFP. A corneal piece from a 16-week-old mouse was mounted flat on a glass slide and photographed under a fluorescence microscope after focused on the top (A), middle (B), and bottom (C) layers of the epithelium. This piece was frozen in embedding compound and serial cryosections of 8-μm thickness were prepared. (D) A cryosection that corresponds to the dotted lines in (A), (B), and (C). Letters at right: corresponding positions of each panel.
Figure 6.
 
A comparison of an en face view (A, B, C) and a cross-sectional view (D) of the same cells with high GFP. A corneal piece from a 16-week-old mouse was mounted flat on a glass slide and photographed under a fluorescence microscope after focused on the top (A), middle (B), and bottom (C) layers of the epithelium. This piece was frozen in embedding compound and serial cryosections of 8-μm thickness were prepared. (D) A cryosection that corresponds to the dotted lines in (A), (B), and (C). Letters at right: corresponding positions of each panel.
Figure 7.
 
A time-lapse sequence of epithelial cell movement at low power (sequence 4-1 in Fig. 10 , Table 1 ). Epithelial GFP fluorescence was recorded with a 4× objective in a living mouse at the indicated times, which represent the age of the mouse. This cornea was tracked for 7 weeks, and images of the last 6 weeks are shown. Arrows: the tip of the bright cluster that was tracked for movement. Traveled distances (Fig. 10 , Table 1 ) were measured in images obtained with a 10× objective (not shown), using limbal capillary markers.
Figure 7.
 
A time-lapse sequence of epithelial cell movement at low power (sequence 4-1 in Fig. 10 , Table 1 ). Epithelial GFP fluorescence was recorded with a 4× objective in a living mouse at the indicated times, which represent the age of the mouse. This cornea was tracked for 7 weeks, and images of the last 6 weeks are shown. Arrows: the tip of the bright cluster that was tracked for movement. Traveled distances (Fig. 10 , Table 1 ) were measured in images obtained with a 10× objective (not shown), using limbal capillary markers.
Figure 8.
 
A time-lapse sequence of epithelial cell movement (sequence 2-1 in Fig. 10 , Table 1 ). Epithelial GFP was recorded in a living mouse, 10 weeks old at the beginning of the recording, over 13 days. Shown are images acquired on days 0 (A, B, C), 4 (D, E, F), 8 (G, H, I), and 13 (J, K, L). At each time point, a low-power image (A, D, G, J), a medium-power image including the limbus (B, E, H, K), and a high-power image of three high-GFP cell clusters (C, F, I, L) are presented. White box: area of the corresponding higher-power images. For medium- and high-power images, overlapping fields were recorded and patched together. The left margin of low- and medium-power images is the limbus, and the central cornea is toward the right. Two white dots at left of the medium power images (B, E, H, K) denote the fixed-position markers as determined by the unique shape of limbal capillary vessels. These vessels appear clearly on a computer monitor after several-fold magnification. (B, E, H, K, arrows) Cells of moderate-GFP cell clusters. The area of the white box in (K) is shown in Figure 9 after fixation of the cornea.
Figure 8.
 
A time-lapse sequence of epithelial cell movement (sequence 2-1 in Fig. 10 , Table 1 ). Epithelial GFP was recorded in a living mouse, 10 weeks old at the beginning of the recording, over 13 days. Shown are images acquired on days 0 (A, B, C), 4 (D, E, F), 8 (G, H, I), and 13 (J, K, L). At each time point, a low-power image (A, D, G, J), a medium-power image including the limbus (B, E, H, K), and a high-power image of three high-GFP cell clusters (C, F, I, L) are presented. White box: area of the corresponding higher-power images. For medium- and high-power images, overlapping fields were recorded and patched together. The left margin of low- and medium-power images is the limbus, and the central cornea is toward the right. Two white dots at left of the medium power images (B, E, H, K) denote the fixed-position markers as determined by the unique shape of limbal capillary vessels. These vessels appear clearly on a computer monitor after several-fold magnification. (B, E, H, K, arrows) Cells of moderate-GFP cell clusters. The area of the white box in (K) is shown in Figure 9 after fixation of the cornea.
Figure 9.
 
Epithelial GFP after fixation of the cornea shown in Figure 8K (white box). The fluorescence microscope was focused on three different levels of epithelium: the superficial (A), suprabasal (B), and basal cell (C) layers, similar to Figure 5 . The focal plane of the brightest cells corresponds to the basal or suprabasal layer of the epithelium.
Figure 9.
 
Epithelial GFP after fixation of the cornea shown in Figure 8K (white box). The fluorescence microscope was focused on three different levels of epithelium: the superficial (A), suprabasal (B), and basal cell (C) layers, similar to Figure 5 . The focal plane of the brightest cells corresponds to the basal or suprabasal layer of the epithelium.
Figure 10.
 
Tracking of GFP-positive epithelial cell clusters in four living mice (seven time-lapse recordings). The labeling, which corresponds to that in Table 1 is, for example, 1-2 denoting mouse 1, sequence 2, and so on. Shown are distances from location markers at the limbus, identified by capillary branches, which were sometimes beyond the margin of the cornea. Also, note that the measurements are of a corneal surface distance, calculated from microscopic images obtained with a 10× objective and do not correspond to the size of the cornea shown in Figure 1 .
Figure 10.
 
Tracking of GFP-positive epithelial cell clusters in four living mice (seven time-lapse recordings). The labeling, which corresponds to that in Table 1 is, for example, 1-2 denoting mouse 1, sequence 2, and so on. Shown are distances from location markers at the limbus, identified by capillary branches, which were sometimes beyond the margin of the cornea. Also, note that the measurements are of a corneal surface distance, calculated from microscopic images obtained with a 10× objective and do not correspond to the size of the cornea shown in Figure 1 .
Table 1.
 
Rate of Centripetal Movement of Corneal Epithelial Cells
Table 1.
 
Rate of Centripetal Movement of Corneal Epithelial Cells
Sequence ID Age (wk) Duration (d) Rate by Total Distance (μm/d) Rate by Regression (μm/d)
1-1 10 18 21.3 22.2
1-2 11 19 31.9 32.1
2-1* 10 13 20.8 22.0
2-2 10 13 21.8 22.3
3-1 7 9 25.6 26.5
4-1, † 7 49 28.0 26.7
4-2, ‡ 11 24 30.4 32.8
Average 25.7 26.4
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