DEVELOPMENTAL DYNAMICS 235:2493–2506, 2006
SPECIAL ISSUE RESEARCH ARTICLE
Collagen Fibril Assembly During Postnatal Development and Dysfunctional Regulation in the Lumican-Deﬁcient Murine Cornea Shukti Chakravarti,1 Guiyun Zhang,2 Inna Chervoneva,3 Luke Roberts,1 and David E. Birk2*
The transparent cornea is the outer barrier of the eye and is its major refractive surface. Development of a functional cornea requires a postnatal maturation phase involving development, growth and organization of the stromal extracellular matrix. Lumican, a leucine-rich proteoglycan, is implicated in regulating assembly of collagen ﬁbrils and the highly organized extracellular matrix essential for corneal transparency. We investigated the regulatory role(s) of lumican in ﬁbril assembly during postnatal corneal development using wild type (Lum⫹/⫹) and lumican-null (Lum⫺/⫺) mice. In Lum⫹/⫹ mice, a regular architecture of small-diameter ﬁbrils is achieved in the anterior stroma by postnatal day 10 (P10), while the posterior stroma takes longer to reach this developmental maturity. Thus, the anterior and the posterior stroma follow distinct developmental timelines and may be under different regulatory mechanisms. In Lum⫺/⫺ mice, it is the posterior stroma where abnormal lateral associations of ﬁbrils and thicker ﬁbrils with irregular contours are evident as early as P10. In contrast, the anterior stroma is minimally perturbed by the absence of lumican. In Lum⫹/⫹ mice, lumican is expressed throughout the developing stroma at P10, with strong expression limited to the posterior stroma in the adult. Therefore, the posterior stroma, which is most vulnerable to lumican-deﬁciency, demonstrates an early developmental defect in ﬁbril structure and architecture in the Lum⫺/⫺ mouse. These defects underlie the reported increased light scattering and opacity detectable in the adult. Our ﬁndings emphasize the early regulation of collagen structure by lumican during postnatal development of the cornea. Developmental Dynamics 235:2493–2506, 2006. © 2006 Wiley-Liss, Inc. Key words: lumican; collagen; cornea; development; ﬁbrillogenesis; extracellular matrix; mouse Accepted 12 May 2006
INTRODUCTION The highly regulated assembly of a stromal matrix containing precisely packed collagen ﬁbrils with uniform, small diameters is a requisite for the development of a transparent, refractive cornea and mechanically stable anterior eye (Maurice, 1957). The structural and functional properties of the adult cornea are acquired in mul-
tiple steps during fetal and postnatal development. These steps and their regulatory mechanisms have not been fully elucidated. It is important to deﬁne these in the mouse model system to understand corneal gene function and to fully utilize the power of knockout models. Toward this end, a recent study explored the growth and acquisition of transparency as the cornea matures between postnatal days 1
and 30 (Song et al., 2003). The decrease in light scattering is almost linear in this time frame as the cornea becomes transparent. At the same time the stroma thickens gradually, except around day 10, just prior to eyelid opening, when there is a sharp increase in thickness. A decline in thickness is seen after eyelid opening that recovers by postnatal day 20. Lumican, a major proteoglycan of the
Department of Medicine, Johns Hopkins University Medical School, Baltimore, Maryland Department of Pathology, Anatomy & Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 3 Department of Pharmacology and Experimental Therapeutics, Thomas Jefferson University, Philadelphia, Pennsylvania Grant Sponsor: National Institutes of Health; Grant numbers: EY05129, EY11654. *Correspondence to: David E. Birk, Department of Pathology, Anatomy & Cell Biology, Thomas Jefferson University, 1020 Locust Street, JAH 543, Philadelphia, PA 19107. E-mail: [email protected]
DOI 10.1002/dvdy.20868 Published online 19 June 2006 in Wiley InterScience (www.interscience.wiley.com).
© 2006 Wiley-Liss, Inc.
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cornea, is critical to the establishment of normal corneal structure and function. In the lumican knockout mouse model, the corneal stroma develops 60% of its normal thickness and fails to acquire wild type transparent properties (Chakravarti et al., 1998, 2000). These studies demonstrate that the sharp increase in stromal thickness observed before eyelid opening in the normal cornea does not occur in lumican-null mice (Song et al., 2003). These earlier studies suggest a prominent regulatory role for lumican in the development and maturation of corneal stromal architecture. Elucidation of the regulatory role(s) of lumican during stromal development is the focus of the current study. Collagen ﬁbril structure and organization are central to corneal structure and transparency. Newly assembled immature collagen ﬁbrils assemble, grow, and mature during postnatal development of the cornea (Birk et al., 1996; Birk, 2001). The corneal keratocyte is the major cell type in the corneal stroma that synthesizes the stromal extracellular matrix (Hamilton, 1972; Beales et al., 1999; Fini, 1999; Chakravarti et al., 2004). It secretes two ﬁbril-forming collagens. Type I collagen is the most prevalent form and type V collagen comprises 10 –20% of the total corneal ﬁbrillar collagen. These collagens co-assemble into heterotypic ﬁbrils (Birk et al., 1988, 1990). The keratocytes also produce at least 4 different small leucinerich proteoglycans: decorin, lumican, keratocan, and osteoglycin (Hassell et al., 1983; Blochberger et al., 1992; Funderburgh et al., 1997; Beales et al., 1999; Dunlevy et al., 2000). Interactions of these proteoglycans with the collagen ﬁbril can modulate ﬁbril structures (Vogel et al., 1984; Rada et al., 1993; Svensson et al., 2000). Therefore, the small leucine-rich proteoglycans are important regulators of matrix assembly. This family of proteoglycans also has been implicated in the regulation of cell growth and behavior; as reservoirs for growth factors; and in cytokine interactions with receptor tyrosine kinases (Ruoslahti and Yamaguchi, 1991; Santra et al., 1997; Moscatello et al., 1998; Iozzo, 1999; Vij et al., 2004, 2005). In addition, in the corneal stroma these proteoglycans, particularly the keratan
sulfate (KS) containing members (lumican, keratocan, and osteoglycin), are important in binding water and, therefore, in regulating stromal hydration, an important parameter in stromal swelling, interﬁbrillar spacing, and corneal transparency (Bettelheim and Plessy, 1975; Doughty, 2001). Each of these four leucine-rich proteoglycans have been “knocked out” in the mouse (Danielson et al., 1997; Chakravarti et al., 1998; Saika et al., 2000; Tasheva et al., 2002; Liu et al., 2003) and all demonstrate abnormal connective tissue phenotypes in various organs and tissues. However, only mice deﬁcient in lumican have a signiﬁcant disruption in corneal collagen ﬁbril architecture. The ﬁbrils in adult lumicannull corneas show a wide range in diameter and contain a population of abnormally large ﬁbrils with irregular contours (Chakravarti et al., 1998, 2000). In addition, alterations in ﬁbril packing are detected by X-ray diffraction analysis (Quantock et al., 2001). The ﬁbril defects in the lumican-null cornea reside primarily in the posterior stroma where lumican content is normally high in the mature wild type mouse (Chakravarti et al., 2000). Decorin-deﬁcient mice have no obvious corneal phenotype (Danielson et al., 1997). Keratocan- and osteoglycin-deﬁcient mice have a modest increase in stromal ﬁbril diameter, but maintain normal circular ﬁbril proﬁles (Tasheva et al., 2002; Liu et al., 2003). In addition, the keratocan-deﬁcient mice demonstrate ﬁbril-packing irregularities and increased interﬁbrillar spacing (Meek et al., 2003). The current study investigates the dynamics of collagen ﬁbril assembly and structure through early postnatal development to the mature adult cornea. Collagen architectural changes that coincide with development and stromal growth in the wild type mouse are contrasted with the defective stromal development in the lumican-null mouse. This study provides essential data addressing collagen assembly and ultrastructure during postnatal stromal development, growth, and maturation of the cornea as well as the regulatory role(s) of lumican in these processes.
RESULTS Postnatal Growth and Thickness of the Corneal Stroma Previous in situ measurements of stromal thickness in living mice demonstrated a signiﬁcantly thinner stroma in Lum⫺/⫺ mice after eye opening (Chakravarti et al., 2000; Song et al., 2003). Our light microscopic analyses of thick sections of the cornea at postnatal day 10, 30, and 90 (P10, P30, P90) conﬁrmed these in vivo results. These data indicate that after P10, the growing (P30) or adult (P90) Lum⫺/⫺ stromas were approximately half as thick as in the Lum⫹/⫹ cornea (Fig. 1, page 2499). These data suggested a structural basis of corneal thinning in the absence of lumican that is preserved after ﬁxation and dehydration rather than one involving changes in hydration.
Corneal Collagen Fibril Structure During Postnatal Development and Maturation Earlier structural studies were done using adult lumican knockout mice and demonstrated defects in collagen ﬁbril structure and organization in the adult posterior stroma (Chakravarti et al., 1998, 2000; Saika et al., 2000). Here, we focused on analyzing the developmental acquisition of this phenotype. Collagen ﬁbrillogenesis is a multistep process, therefore, elucidation of the period in development when the defect ﬁrst manifests itself will provide mechanistic insight into the collagen assembly steps that are altered in Lum⫺/⫺ mice. The murine cornea begins to acquire optical transparency after P10; this coincides with an increased matrix organization in the developing cornea seen as a gradual decrease in ﬁbril spacing and an associated decreased backscattering of light (Quantock et al., 2001; Song et al., 2003). These processes involve the assembly of small-diameter collagen ﬁbrils with a narrow diameter distribution. Fibril structure was analyzed in the stroma during postnatal development (P10, P30) and at maturity (P90) (Fig. 2). In the Lum⫹/⫹ mice, no obvious anteriorposterior difference in collagen ﬁbril
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structure was observed. In contrast, in the Lum⫺/⫺ mice, anterior-posterior differences in ﬁbril structure were acquired with postnatal development. In P10 Lum⫺/⫺ mice, the anterior stroma contained ﬁbrils with relatively normal structures. At P30, the anterior stroma contained occasional thick ﬁbrils and small irregularities in the ﬁbril contour (data not shown), which became more pronounced by P90 (Fig. 2A). Collagen ﬁbril structure was analyzed in the posterior stroma of postnatal corneas. In Lum⫹/⫹ corneas, small-diameter ﬁbrils with circular proﬁles were assembled throughout postnatal development, growth, and aging. Morphologically the ﬁbril structures at different stages were indistinguishable. However, ﬁbril packing increased in regularity from P10 to P90. In the Lum⫺/⫺ mice, ﬁbril architecture was distinct from that seen in the Lum⫹/⫹ posterior stromas at these time points (Fig. 2B). Our previous study of the Lum⫺/⫺ mouse demonstrated an increased ﬁbril diameter and irregular contoured ﬁbrils in the mature 7.5month cornea (Chakravarti et al., 2000). The current analyses of the development of aberrant collagen ﬁbrils demonstrated the early appearance of collagen anomalies, namely fused ﬁbrils and ﬁbrils with abnormally large diameters as well as altered diameter distributions by P10. By 1 month of develop-
Fig. 2. Spatial and temporal acquisition of abnormal collagen ﬁbril structure in the Lum⫺/⫺ corneal stroma. Ultrastructural analyses of ﬁbril structure were done in the anterior and posterior stroma during early postnatal development (P10), growth (P30), and in mature adult (P90) corneas. A: Fibril structure and organization in the anterior stroma were comparable in Lum⫹/⫹ and Lum⫺/⫺ mice. However, at P90, occasional large-diameter ﬁbrils (arrow) were present in the Lum⫺/⫺ corneas. B: Fibril architecture was markedly altered in the posterior stroma at all stages of corneal development, in the Lum⫺/⫺ mouse, with the frequency of structurally aberrant ﬁbrils increased from P10 to P90. In the P10 Lum⫺/⫺ mouse cornea, abnormal ﬁbrils, consisting of large ﬁbrils and small clusters of laterally associated ﬁbrils (arrows), were evident; at P30 and P90, large structurally aberrant ﬁbrils with irregular cross-sectional proﬁles accumulated, with an increased incidence of laterally associated ﬁbrils. At all 3 stages, the packing of the posterior stroma also was disrupted in the Lum⫺/⫺ corneas compared to the Lum⫹/⫹ controls. P10, postnatal day 10; P30, 1 month; and P90, 3 months.
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ment (P30), a population of largediameter ﬁbrils was consistently observed in the posterior stroma, with virtually all regions of the posterior stroma containing abnormal ﬁbrils. At P90, the abnormal ﬁbrils were larger and more irregular in proﬁle than those seen earlier in development, with increased incidence of abnormal associations of collagen ﬁbrils. After 3 months, the posterior stromal phenotype was relatively stable with no detectable changes between P90 and 1 year (data not shown).
Spatial (Anterior-Posterior) Differences in Stromal Fibrillogenesis Distinct structural, biochemical, and functional differences have been demonstrated along the anterior-posterior axis in human, rabbit, and bovine corneas (Bettelheim and Goetz, 1976; Castoro et al., 1988; Freund et al., 1995). Therefore, development of the stromal matrix in the anterior and posterior stroma of the mouse was deﬁned in Lum⫹/⫹ and Lum⫺/⫺ mice at P10, P30, and P90. No obvious anterior-to-posterior differences in collagen ﬁbril structure were observed in Lum⫹/⫹ mice, while Lum⫺/⫺ mice acquired a posterior enrichment of structural defects. The developmental and spatial acquisition of these defects were further characterized using analyses of ﬁbril diameters. In both Lum⫹/⫹ and Lum⫺/⫺ mice, the mean ﬁbril diameters were larger in the posterior versus anterior stroma at all developmental stages studied (Fig. 3). Furthermore, the diameter distributions in the posterior stroma of the Lum⫺/⫺ mice contained a distinct righthand shoulder, consistent with the presence of two ﬁbril subpopulations: one population of relatively normal ﬁbrils with circular proﬁles and a second of large diameter ﬁbrils with irregular proﬁles. A statistical analysis of median diameters in the 3 combined developmental stages for Lum⫹/⫹ versus Lum⫺/⫺ mice was done. The median ﬁbril diameters in the posterior stroma were an average of 4.1 nm larger than in the anterior stroma in Lum⫹/⫹ mice (P ⬍ 0.001, 95% CI: 3.3, 4.9) and 3.0 nm larger in the posterior stroma of Lum⫺/⫺ mice (P ⬍ 0.001, 95% CI: 2.2, 3.7).
Fig. 3. Altered collagen ﬁbril diameter distributions in the Lum⫺/⫺ corneal stroma. Lum⫹/⫹ mice had small diameter ﬁbrils within a narrow range in both the (A) anterior and (B) posterior stroma with the mean ﬁbril diameters consistently larger in the posterior stroma. A: In the anterior stroma, the distribution of ﬁbril diameters in the Lum⫺/⫺ corneas was comparable to that observed in the Lum⫹/⫹ corneas, but the spread was somewhat greater at P30 and P90, with no major shoulder corresponding to large, irregular diameter ﬁbrils observed. B: In the posterior stroma, Lum⫺/⫺ mice demonstrated a distinct shoulder, indicative of larger-diameter ﬁbrils. This shoulder was present at all 3 stages examined. The number of large, abnormal ﬁbrils increased with postnatal development. Values are means ⫾ sd (n) and the diameter range (min-max). P10, postnatal day 10; P30, 1 month; and P90, 3 months.
Fibril Assembly Is Altered in the Lumⴚ/ⴚ Mouse Two distinct ﬁbril subpopulations, one relatively normal-appearing and a second composed of abnormal, laterally fused ﬁbrils, were assembled in the Lum⫺/⫺ cornea. A dysfunctional regulation of ﬁbril growth associated with the absence of lumican presumably resulted in the aberrant, laterally associated subpopulation of ﬁbrils. To determine if the normal-appearing ﬁbril subpopulation in the Lum⫺/⫺ mice was altered compared to the ﬁbrils in the wild type corneas, the two subpopulations were analyzed separately in Lum⫹/⫹ and Lum⫺/⫺ mice. The dominant, normal-appearing ﬁbril subpopulation was analyzed using data sets where the large structurally aberrant ﬁbril subpopulation was
removed. As described in the Experimental Procedures section, the structurally aberrant ﬁbrils were identiﬁed using an established outlier detection rule (Hoaglin et al., 1986). The dominant, cylindrical ﬁbril subpopulations had a symmetric distribution of ﬁbril diameters. No signiﬁcant differences in median ﬁbril diameter were observed between Lum⫺/⫺ and Lum⫹/⫹ mice and the difference among ages (P10, P30, P90) was only marginally signiﬁcant (P ⬍ 0.058). The spread and heterogeneity of the ﬁbril diameters in the cylindrical ﬁbril subpopulation were characterized by analyses of sample inter-quartile ranges (Q3– Q1), as described in the Experimental Procedures section. The inter-quartile range encompasses the central 50% of the dominant ﬁbril subpopulation and
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Fig. 4. Diameter heterogeneity in the structurally normal ﬁbril subpopulation in the Lum⫺/⫺ corneal stroma. Analysis of the dominant, structurally normal ﬁbril subpopulation in Lum⫺/⫺ and Lum⫹/⫹ stromas during postnatal development. Lum⫺/⫺ mice (solid circles) assembled ﬁbrils with a significantly broader distribution compared to Lum⫹/⫹ mice (open circles). The difference between Lum⫺/⫺ and Lum⫹/⫹ cylindrical subpopulations was signiﬁcant at P30 and P90 in both the (A) anterior and (B) posterior stroma. Means of inter-quartile ranges (minimum and maximum values for central 50% of the ﬁbril diameters) and their 95% conﬁdence intervals are shown. For these analyses, the data sets were stripped of the larger, structurally abnormal ﬁbrils (outliers) as described in the Experimental Procedures section.
is used to measure the spread of the data that does not necessarily follow a normal distribution. For a normal distribution the inter-quartile range is the same as the interval (mean ⫾ 0.67 sd) and the length of such inter-quartile range is 1.35 ⫻ sd. A signiﬁcantly broader distribution of ﬁbril diameters was observed in the Lum⫺/⫺ versus Lum⫹/⫹ mice as determined from analyses of the inter-quartile ranges (Fig. 4). This broadening of the distribution indicated signiﬁcant differences in diameter heterogeneity between Lum⫺/⫺ and Lum⫹/⫹ distributions (P ⬍ 0.001), between different age groups (P ⬍ 0.001), and between the anterior and posterior stroma (P ⫽ 0.013). Moreover, the age-by-genotype interaction was signiﬁcant (P ⬍ 0.001), indicating that changes in diameter variability associated with age had different patterns in Lum⫹/⫹ and Lum⫺/⫺ animals (Fig. 4). At P10, the diameter ranges were similar for all genotypes and locations. At P30 and P90, diameter heterogeneity was signiﬁcantly greater (P ⬍ 0.001) in Lum⫺/⫺ compared to Lum⫹/⫹ mice in both the anterior and posterior stroma. In addition, at P90, diameter heterogeneity in the posterior stroma was greater than in the anterior
stroma of Lum⫺/⫺ animals (P ⫽ 0.004), but not in the Lum⫹/⫹ mice (Fig. 4). The large, aberrant ﬁbril subpopulation also was analyzed separately (Table 1). These analyses indicated a signiﬁcant correlation between the presence of aberrant ﬁbrils and the absence of lumican. With postnatal development and maturation of the corneal stroma, there was a signiﬁcant increase in the number of large, aberrant ﬁbrils (outliers). Aberrant ﬁbrils were identiﬁed as outliers using standard statistical criteria as described in the Experimental Procedures section. In the posterior stroma where the large, abnormal ﬁbrils predominate, a total of 204 outliers, including 44 extreme outliers, were identiﬁed. All extreme outliers, except one, were found in Lum⫺/⫺ groups. These include 4 extreme outliers at P10 (0.2% incidence rate), 13 extreme outliers at P30 (0.6% incidence rate), and 26 extreme outliers at P90 (1.2% incidence rate). These results were analyzed by looking at all the data using a linear mixed effects model and asking what the chances are of ﬁnding abnormal ﬁbrils. For the number of all posterior outliers, the generalized linear mixed effects model provided a good ﬁt and predicted a total of 204.8
outliers. Signiﬁcant differences in numbers of abnormal ﬁbrils between Lum⫺/⫺ and Lum⫹/⫹ posterior stromas were observed in each of the age groups analyzed (Table 1). The Lum⫺/⫺ mice were more likely than Lum⫹/⫹ animals to have abnormally large-diameter ﬁbrils, about 2.5 times at P10, 4.8 times at P30, and 4.0 times at P90. A signiﬁcant increase in large, aberrant ﬁbrils (outliers) was seen between P10 and both P30 and P90 in the Lum⫺/⫺ stromas (Table 1). In the Lum⫺/⫺ animals, the incidence rate of outliers was 1.8 times higher at P30 and 2.1 times higher at P90 as compared to P10. In the Lum⫹/⫹ posterior stroma, no signiﬁcant differences in incidence rates of outliers were observed among different age groups. In the anterior stroma, only 37 outliers were identiﬁed in the entire data set and only one extreme outlier was observed. For the counts of all anterior outliers, the generalized linear mixed effects model provided a good ﬁt and predicted a total of 37.4 outliers. Consistent with our experimental analyses, this model yielded no signiﬁcant differences between Lum⫹/⫹ versus Lum⫺/⫺ or among developmental stages in the anterior stroma.
Lumican Localization During Postnatal Development of the Cornea Our previous study demonstrated increased localization of lumican in the posterior stroma of the adult mouse cornea (Chakravarti et al., 2000). Here, we addressed the developmental acquisition of this restricted spatial localization pattern and the relationship to structural defects in the developing Lum⫺/⫺ stroma (Fig. 5). At P10, lumican was present homogeneously throughout the corneal stroma. By P45, lumican was localized almost exclusively in the posterior stroma. The posterior expression pattern was maintained in the mature (P90) cornea with some focal, heterogeneous expression in the anterior stroma of mature mice. Thus, the pronounced posterior ﬁbril phenotype present in the Lum⫺/⫺ cornea coincides with the developmental pattern of lumican deposition in the Lum⫹/⫹ cornea. Lumican is one of ﬁve small leucine-
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TABLE 1. Analyses of the Number of Large, Abnormal Fibrils in the Posterior Stroma of Lumⴚ/ⴚ and Lumⴙ/ⴙ Corneas Incidence of large outliers in the posterior stroma Genotype
Observed incidence rate (%)
Standard error of incidence rate (%)
Lum⫺/⫺ Lum⫺/⫺ Lum⫺/⫺ Lum⫹/⫹ Lum⫹/⫹ Lum⫹/⫹
P10 P30 P90 P10 P30 P90
2,324 2,065 2,192 1,747 3,021 2,434
34 55 67 10 17 21
1.46 2.66 3.06 0.57 0.56 0.86
0.25 0.35 0.37 0.18 0.14 0.19
Incidence rate ratiob
Lum⫺/⫺ vs. Lum⫹/⫹ Lum⫺/⫺ vs. Lum⫹/⫹ Lum⫺/⫺ vs. Lum⫹/⫹ Lum⫺/⫺ Lum⫺/⫺ Lum⫺/⫺
P10 P30 P90 P30 vs. P10 P90 vs. P10 P90 vs. P30
2.54 4.79 3.99 1.84 2.05 1.12
95% Conﬁdence interval limits Lower
1.19 2.62 2.17 1.12 1.27 0.73
5.46 8.77 7.32 3.01 3.32 1.71
0.017 ⬍0.001 ⬍0.001 0.015 0.003 0.615
P10, postnatal day 10; P30, 1 month; P90, 3 months. The incidence rate ratios were estimated from the ﬁtted generalized linear mixed effects model, which accounts for correlation of data from the same animals and negatives. Since adjustments were made for these correlations, the estimates are close, but not exactly equal to the ratios of corresponding incidence rates reported.
rich proteoglycans in the corneal stroma. To evaluate the role of lumican in the corneal stroma in context, the localization of the other small leucine-rich proteoglycans was ana-
lyzed over the same postnatal period (Fig. 6). Keratocan demonstrated a homogeneous localization throughout the mature (P90) stroma. Decorin and biglycan both demonstrated a compa-
rable homogeneous stromal localization at P90. In contrast, osteoglycin was localized primarily to the epithelium and epithelial basement membrane zone with lower immuno-reac-
Fig. 5. Temporal and spatial distribution of lumican in the Lum⫹/⫹ cornea. Immunoﬂuorescence micrographs showing the distribution of Lum⫹/⫹ corneas at P10, P45, and P90. At P10, there was a homogeneous distribution of lumican reactivity throughout the corneal stroma. At P45, the reactivity was primarily in the posterior stroma. However, the anterior stroma had anti-lumican reactivity above that observed in the Lum⫺/⫺ control corneas. In the mature P90 cornea, the posterior stroma retained the higher level of lumican expression seen at P45. However, the anterior stroma showed heterogeneous regions with increased lumican reactivity. As expected, reactivity for lumican was not observed in the Lum⫺/⫺ corneas. Fig. 6. Small leucine-rich proteoglycan localization during postnatal corneal development. Localization of keratocan, osteoglycin, decorin, and biglycan in P90 corneas using immunoﬂuorescence microscopy. Keratocan demonstrated a homogeneous localization throughout the mature corneal stroma. No reactivity was observed in the epithelium or endothelium. Both decorin and biglycan demonstrated a comparable homogenous localization throughout the stroma, with biglycan reactivity substantially weaker than decorin. In contrast, osteoglycin localized to the epithelium and basement membrane zone (arrowhead). In addition, reactivity was associated with keratocytes and the endothelium. Arrowheads show osteoglycin reactivity in epithelium and basement membrane. Nuclei were counterstained with DAPI. Primary antibody was omitted in the negative controls. Arrows, endothelium; Ep, epithelium. Fig. 9. Lumican in the regulation of corneal stromal collagen ﬁbril formation. Schematic diagram presenting our model for the role of lumican in the regulation of collagen ﬁbril assembly and growth. Collagen molecules assemble into ﬁbril intermediates in close association with the keratocyte surface. In most connective tissues, these preformed ﬁbril intermediates then undergo end-to-end linear growth and lateral associations leading to larger-diameter ﬁbrils. In the cornea, only the linear growth takes place during normal development. However, during postnatal corneal development in Lum⫺/⫺ mice, lateral growth occurs in a limited ﬁbril subpopulation with restricted spatial and temporal distribution. A: In Lum⫹/⫹ mice, lumican (blue circles) associates with the shaft of newly assembled ﬁbril intermediates. This interaction stabilizes the ﬁbril intermediates and prevents lateral ﬁbril growth as the intermediates grow linearly. B: In Lum⫺/⫺ mice, in the absence of lumican, there is no stabilizing interaction and the immature ﬁbril intermediates laterally and linearly associate. This unregulated growth yields abnormally large-diameter ﬁbrils and laterally associated ﬁbrils with irregular cross-sectional ﬁbril proﬁles. However, the presence of normal corneal ﬁbrils in the anterior stroma as well as intermixed with abnormal ﬁbrils in the posterior stroma indicates that there are other regulatory interactions acting during postnatal development of the corneal stroma. The other closely related small leucine-rich proteoglycans, speciﬁcally keratocan, decorin, and biglycan, are potential candidate regulatory molecules as well as ﬁbril-associated collagens type XII and XIV found with restricted temporal and spatial expression patterns during this period.
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Fig. 1. Stromal thickness is reduced in growing and mature Lum⫺/⫺ corneas. Light microscopy of thick sections (1 m) of the cornea stained with methylene blue-Azur B. Compared to Lum⫹/⫹ mice, Lum⫺/⫺ mice had a consistently thinner stroma (S) at 1 month (P30), a phenotype that was maintained in the mature 3-month-old (P90) Lum⫺/⫺ corneas. Arrowheads indicate the boundaries of the stroma.
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tivity associated with the keratocytes and endothelium. Unlike lumican, the spatial localization of keratocan, osteoglycin, decorin, and biglycan did not change during postnatal development studied at P10, P30, and P90 (data not shown). Semi-quantitative RT-PCR of total RNA with lumican speciﬁc primers indicated high lumican levels at P4 and P10 with a 30 – 45% decrease by 3– 4 weeks. This expression pattern remained relatively stable at this level thereafter (Fig. 7A). However, there was a consistent small increase at 3 months coinciding with the period where the spatial localization of lumican within the stroma changes. Immuno-blotting of total protein extract from the cornea with an anti-lumican polyclonal antibody demonstrated high levels of the lumican core protein at P4 and P8 with decreased expression by P10 and P12 (Fig. 7B). The high lumican content during the early stages of postnatal development actually preceded the stage when an abnormal ﬁbril phenotype appeared in the Lum⫺/⫺ mice. This suggested that high initial levels of lumican were important in the regulation of stromal ﬁbril assembly. At P10 and P12, there was a decrease in faster migrating bands. In general, the cornea is known to gain KS chains as it matures. We interpreted the faster bands at the younger stages as immature core proteins with few KS-side chains. The shift from fast to slower migrating bands is less likely to be due to a shift in the length of the KS-side chains, which in the mouse are short to begin with. The faster migrating bands were not observed, at the later stage, because with development these immature core proteins became modiﬁed by attachment of KS-chains and appeared as the slower bands. One would then expect to see increased intensity of the slower bands at P10 and P12, but we instead saw a decrease. We interpret this to mean that less core protein was made at the later stages and so lower amounts of the KS-modiﬁed proteoglycans were observed. This also followed the pattern of reduced lumican mRNA levels at these later stages. We do not believe that the reduced reactivity at P10 and P12 was due to reduced antibody penetration and staining of the core pro-
Fig. 7. Lumican content during postnatal corneal development. A: Lumican mRNA was analyzed using semi-quantitative PCR. Lumican mRNA was highest at postnatal day 4 (P4) and P10. There was a decrease to 3 weeks (3W) and relatively stable expression thereafter. Lumican was ampliﬁed from total RNA extracts of the cornea. The amount of lumican amplicon was assessed relative to 18S RNA, with the relative amount at P4 set at 1.0. Error bars represent the standard deviation. B: Lumican core protein content was studied using semi-quantitative immuno-blot analyses. The amount of lumican core protein was assessed by immunoblotting total protein extracts from the cornea at P4, P8, P10, and P12.
tein in the fully glycosaminoglycanated form, because this antibody, characterized in the past, has not shown reduced staining of mature proteoglycan versus deglycosylated core protein (Chakravarti et al., 1998). We also investigated mRNA levels of the other corneal proteoglycans; keratocan, osteoglycin, decorin, and biglycan at these postnatal developmental stages (Fig. 8). Keratocan and decorin mRNA levels were constant throughout the developing stages analyzed. Biglycan and osteoglycin mRNA was high at P4 and P10, reduced by 3 weeks, after which the expression levels remained constant.
DISCUSSION The cornea consists of a stratiﬁed, multilayered epithelium, a stroma, and a single layered endothelium that by virtue of its ion pump regulates stromal hydration. Although fully formed at birth, the cornea undergoes considerable, yet poorly deﬁned preand postnatal development, growth, and maturation. During chicken development, the corneal stroma becomes compacted before hatching by dehydration and changes in ﬁbril packing (Hay, 1980; Connon et al., 2004). In the mouse, there is an initial decrease in thickness when endothe-
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Fig. 8. Small leucine-rich proteoglycan mRNA during postnatal development of the mouse cornea. A: Keratocan and osteoglycin mRNAs were analyzed using semi-quantitative PCR. Keratocan mRNA was constant during the period studied while osteoglycin mRNA was highest at P4 and 10 followed by a decrease and stable levels from 3W to 6M⫹. B: Decorin mRNA levels were constant during the period analyzed. In contrast, biglycan levels were highest at P4, decreased to 3W, and were stable thereafter. Small leucine-rich proteoglycan mRNAs were ampliﬁed from total RNA extracts of the cornea. The amount of product was assessed relative to 18S RNA at postnatal day (P) 4 and, 3, 4, 6, and 8 weeks (W) as wells as 3 months (3M) and greater than 6 months (6M⫹). C: The amount of amplicon was assessed relative to 18S RNA, with the relative amount at P4 set at 1.0 for keratocan, osteoglycin, decorin, and biglycan. Error bars represent the standard deviation.
lial pump-function ensues after eyelid-opening. Thereafter, the stroma thickens gradually until P30 (Song et al., 2003). In larger animals, such as the cat, the cornea grows for up to 2 years after birth (Moodie et al., 2001) and in the dog for up to the ﬁrst 8 months (Montiani-Ferreira et al., 2003). In the human, eyelid opening– associated changes are prenatal, but the cornea grows and increases in thickness after birth up to 3 years of age. Therefore, the manifestation of many genetic defects during these early formative stages of the cornea is not unexpected. At maturity the fully functioning cornea, of which 90% is made up of collagenous stroma, provides three quarters of the refractive power of the eye (Pepose and Ubels, 1992). Collagen ﬁbrils and the anionic collagen-binding proteoglycans are organized into sheets or lamellae and form the bulk of the stroma. Adaptive changes in the ultra-
structure of the stroma accompany the structural changes that occur before and after birth and are critical to the acquisition of corneal transparency. Here we investigated the assembly and maturation of collagen ﬁbrils during postnatal growth and development of the cornea in mice homozygous for the wild type allele or genetargeted null allele of lumican. The data indicate a difference in ﬁbril morphology and organization along the anterior-posterior axis, as noted in the chicken, rabbit, and human cornea (Bettelheim and Goetz, 1976; Castoro et al., 1988; Freund et al., 1995). In the Lum⫺/⫺ stroma, ﬁbril structural defects arose early, were somewhat progressive, and primarily affected the posterior stroma where lumican expression became localized during normal corneal maturation (⬃6 weeks). First, our comparison of the anterior and posterior stroma at postnatal
P10, P30, and P90 with respect to collagen ﬁbril diameter revealed a small, but signiﬁcant increase in ﬁbril diameter from anterior to posterior stroma of the Lum⫹/⫹ mouse across all ages studied. In the human and rabbit, on average ﬁbril diameters were smaller in the posterior stroma. In the human and rabbit studies, the current study examined very young to young adult mouse corneas. The observed difference in ﬁbril diameter between the posterior and the anterior stroma may be simply age related. Alternatively, it may be species-speciﬁc with the extremely thin mouse cornea regulating collagen ﬁbril assembly in a manner that is different from other larger species. Across species, the overall organization and packing of collagen ﬁbrils was consistently more regular in the posterior stroma. Our data, from the P10, P30, and P90 mouse cornea, suggest ﬁbril organization in the posterior stroma increased at the later stage. A recent study of the developing chicken cornea showed that at the early stages, the anterior stroma was far more organized with a higher density of collagen ﬁbrils than the posterior stroma. Fibril density increased in an age-dependent manner in the posterior stroma, ultimately catching up with the anterior stroma in collagen organization (Connon et al., 2004). In the human cornea, the anterior stroma was shown to be largely responsible for establishing corneal curvature. It was resistant to swelling and structurally more stable with the lamellae of collagen ﬁbrils more interwoven than those in the posterior stroma (Muller et al., 2001). Presumably, these differences were modulated by various compositional differences along the anterior-posterior axis of the cornea. For example, the water content increased in the bovine cornea from the epithelium towards the corneal interior, alongside differences in the glycosaminoglycan content. The anterior stroma was rich in chondroitin sulfate, while the posterior stroma was rich in keratan sulfate (KS) which has greater water retentive power (Bettelheim and Goetz, 1976; Castoro et al., 1988). Ample studies link keratan sulfate with the transparent refractive properties of the corneal stroma. Deﬁciencies in
2502 CHAKRAVARTI ET AL.
keratan sulfate metabolism were linked to macular corneal dystrophies (Hassell et al., 1980; Niel et al., 2003). During acquisition of corneal transparency, the developing cornea gains in KS, conversely wounding and loss of transparency, was associated with a decrease in KS (Funderburgh, 2000). Thus, lumican, the most abundant keratan sulfate– containing proteoglycan of the corneal stroma is likely to be a key regulator of corneal transparency. In this study, we demonstrated temporal as well as spatial differences in lumican expression in the developing mouse cornea. In the early neonatal stage (P10), there was a uniform distribution of lumican across the stroma. By 1 month, lumican began to show a preferential localization to the posterior stroma. Quantitative assessment of lumican in the mouse cornea indicated high levels of the lumican core protein during the early postnatal stages (P4, P8) with a substantial decrease observed by P10. At the mRNA level, we also noted high expression at P4 and P10. This was down-regulated by 3 weeks postnatal. Our ﬁndings on lumican distribution are in agreement with the temporal and spatial distribution of KS in the cornea and the general consensus that this proteoglycan plays a key role in the development and maintenance of corneal transparency. We know that lumican regulates collagen ﬁbril diameter and organization in the adult cornea (Chakravarti et al., 1998; 2000; Saika et al., 2000). The spatial distribution of collagen ﬁbrillar ultrastructural anomalies coincides with the posterior-rich distribution of lumican in the normal cornea, with transparency defects localized to the posterior stroma of the Lum⫺/⫺ cornea. Given the temporal and spatial distribution of lumican, how is the structure and function of the cornea affected during its postnatal development and maturation in the Lum⫺/⫺ mice? In our earliest study, corneal opacity was visible by slit lamp in the adult six-week-old animals (Chakravarti et al., 1998). In vivo confocal microscopy of the developing Lum⫺/⫺ cornea indicated light scattering defects by three weeks (Chakravarti et al., 2000; Song et al., 2003). These functional anomalies were corroborated by the current iden-
tiﬁcation of detectable structural anomalies at the early stages. The ultrastructural analyses of the postnatal cornea presented here demonstrated occasional abnormally associated ﬁbrils and an increased range in the collagen ﬁbril diameter distribution by P10. The number of abnormal ﬁbrils and the large-diameter ﬁbrils increased to maturity when the phenotype stabilized, arguing against a major regulatory role for lumican in the earliest, molecular assembly stages of ﬁbrillogenesis. Lumican-deﬁciency was associated with lateral ﬁbril growth in the posterior stroma. Lateral growth is an important step in ﬁbrillogenesis in virtually all soft connective tissues. However, it is inconsistent with corneal transparency. In other tissues, i.e., sclera, tendon, the down regulation of lumican expression was associated with the onset of ﬁbril growth (Ezura et al., 2000; Chakravarti et al., 2003). This is congruent with a key role for lumican-ﬁbril interactions in the regulation of initiation of lateral ﬁbril growth. We propose a model (Fig. 9, page 2499) where interactions with lumican stabilize the immature ﬁbril intermediate and thereby restrict lateral growth. We hypothesize that these interactions are permissive for linear ﬁbril growth since linear growth is required and occurs during stromal development (Birk et al., 1996). In addition, the initiation of ﬁbril assembly does not appear to involve lumican since our data demonstrated no structural changes until after eye opening. This early step has been shown to be regulated by type I/V collagen interactions (Wenstrup et al., 2004; Segev et al., 2006). The absence of interactions between lumican and the ﬁbrils lead to a dysfunctional regulation of ﬁbrillogenesis, particularly in the posterior stroma. This allows abnormal lateral association and partial ﬁbril fusion generating the structurally aberrant ﬁbrils seen in the Lum⫺/⫺ cornea. The spatial and temporal patterns in the lumican-deﬁcient phenotype suggest that other regulatory interactions are involved in the coordinate regulation of stromal ﬁbrillogenesis. Other small leucine-rich proteoglycans are good candidates. Interestingly, genetargeted mice deﬁcient in the other leucine-rich proteoglycans— osteoglycin
(Tasheva et al., 2002), keratocan (Liu et al., 2003), and decorin (Danielson et al., 1997)— have milder corneal phenotypes with respect to collagen architecture and no disruption in transparency. However, these leucine-rich proteoglycans may interact with lumican in the regulation of collagen ﬁbrillogenesis. The interactions of the closely related class I versus class II leucine-rich proteoglycans in the regulation of ﬁbrillogenesis have been described (Ezura et al., 2000; Ameye and Young, 2002; Chakravarti et al., 2003). Lumican and keratocan are class II leucine-rich proteoglycans and lumican has been shown to regulate corneal keratocan synthesis (Carlson et al., 2005). Our demonstration of a predominantly cellular localization of osteoglycin during postnatal corneal development suggests that this proteoglycan is not directly involved. The anterior-posterior differences in the regulation of ﬁbrillogenesis may be associated with differences in the composition and stoichiometry of other ﬁbril-associated matrix macromolecules such as collagens type XII and XIV, which have demonstrated to have changing patterns during corneal development (Anderson et al., 2000; Young et al., 2002; Marchant et al., 2002). In summary, our study demonstrates that in the Lum⫺/⫺ mouse, the posterior stroma acquires collagen ﬁbril structural disruptions by P10, while the anterior stroma is relatively unperturbed at this early stage. The posterior collagen ﬁbril anomaly stabilizes in the mature adult, at which point the anterior stroma shows subtle structural defects. Our ﬁndings reinforce the concept that in the corneal stroma, the anterior and posterior regions have different mechanisms of development and maturation. Expression and accumulation of lumican early in corneal development may be essential for the maturation of collagen ﬁbrils and associated with a properly hydrated and transparent cornea. After the early framework is established, the anterior stroma, primarily responsible for deﬁning corneal curvature, may be under the control of other factors/ interactions that are unaffected by lumican-deﬁciency.
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TABLE 2. Semi-Quantitative PCR
Lum-F Lum-R Ogn-F Ogn-R Kera-F Kera-R Dcn-F Dcn-R Bgn-F Bgn-R
5⬘-GTC 5⬘-ATC 5⬘-TGC 5⬘-GAA 5⬘-CTT 5⬘-GAT 5⬘-AGG 5⬘-CCG 5⬘-GAC 5⬘-GTG
ACA TTG TTT GCT TCC CGG CAT CCC AAC GTC
GAC GAG GTG GCA CCG TGG TCA AGT CGT CAG
CTG TAA GTC CAC AAT CTT AAC TCT ATC GTG
EXPERIMENTAL PROCEDURES Animals Gene targeted mice deﬁcient in lumican (lumtm1sc/lumtm1sc), here referred to as Lum⫺/⫺, in an outbred CD-1 background (Chakravarti et al., 1998) and wild-type mice (Lum⫹/⫹) in the same genetic background were used in these experiments between postnatal day 4 and 12 months. All animal studies were performed in compliance with IACUC approved animal protocols.
Immunoﬂuorescence Microscopy Corneas were ﬁxed with 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.3) for 30 min on ice as previously described (Ezura et al., 2000; Chakravarti et al., 2000).The tissues were cryoprotected with 2 M sucrose-PBS, and frozen in optimal cutting temperature compound (OCT; Tissue Tek, Miles Laboratories, Naperville, IL). Sections (6 m) were cut and mounted on poly-L-lysine– coated slides, reduced with sodium borohydride, and nonspeciﬁc binding sites were blocked by incubation in 5% bovine serum albumin (BSA) in PBS overnight at 4° C. Sections were then incubated with rabbit anti-mouse polyclonal antibodies speciﬁc for the protein cores of lumican, keratocan (Chakravarti et al., 2000), decorin (LF113), biglycan (LF159) (Fisher et al., 1995), or osteoglycin (Ge et al., 2004). The secondary antibodies were goat anti-rabbit IgG dichlorotriazynyl amino ﬂuorescein– conjugated at 1:150 (Jackson ImmunoResearch,
CAG GAC ACA AGC CAA GAT CTC ATG CGC AAG
TGG AGT TGG ACA TGC TTC TCG ACA AAA TTC
CTC AT-3⬘ GGT CC-3⬘ AT-3⬘ AT-3⬘ TA-3⬘ AT-3⬘ TG-3⬘ AG-3⬘ GT-3⬘ GT-3⬘
Amplicon size (bp)
Optimal cycle no.
West Grove, PA) or goat anti-rabbit IgG Alexa Fluor 568 or 488 at 1:400 (Molecular Probes, Eugene, OR). Each primary antibody was serially diluted from 1:50 to determine appropriate dilutions in the linear range for experimental analyses. The following primary antibody dilutions were used: anti-lumican 1:200; anti-osteoglycin 1:100; anti-keratocan 1:200; antidecorin 1:200; and anti-biglycan 1:100. Negative control samples were incubated identically, except the primary antibody was excluded. To visualize nuclei, the slides were mounted in glycerol solution with 1 g/ml Hoechst stain. Images were captured using a digital camera (Optronics, Goletta, CA), with set integration times and identical conditions to facilitate comparisons between samples.
Western Blots Total protein was extracted from homogenized corneas using M-PER mammalian protein extraction reagent (Pierce Technology) at room temperature for 5 min, followed by adding the Halt protease inhibitor mixture (Pierce Technology). The proteins were resolved by SDS-PAGE in an 8% polyacrylamide gel and transferred to 0.2-m pore size nitrocellulose membrane (Invitrogen) for immunoblotting. Lumican was detected using a polyclonal antibody against mouse lumican described above (Chakravarti et al., 2000). Actin was immunostained with a polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) as a control for equivalent loading. Anti-rabbit (1: 1,000) and anti-goat (1:2,500) HRP
conjugated secondary antibodies were used with the appropriate primary antibodies. Membranes were developed using Super Signal West Pico, stable peroxide solution, and Luminol/Enhancer (1:1 ratio).
Semi-Quantitative PCR Semi-quantitative RT-PCR analyses were done as previously described (Ezura et al., 2000; Young et al., 2002; Zhang et al., 2003). Primer sequences for lumican, keratocan, osteoglycin, decorin, and biglycan are given in Table 2. Classic II 18S internal standard (Ambion) was used as reference gene yielding a 324-bp product. The amplicon size; primer pair to competimer ratio yielding comparable product intensities for 18S and each proteoglycan; and the optimal PCR cycle number are also presented in Table 2. PCR programming was: 94°C 2 min followed by cycles of 94°C 20 sec, 60°C 20 sec, 72°C 40 sec, and a ﬁnal extension at 72°C for 10 min. For each age, PCR was done with at least 6 different cDNA preparations from at least 2 independent mRNA samples. The mean density of proteoglycan bands was normalized to the 18S reference gene and presented as mean density ⫾ standard deviation.
Transmission Electron Microscopy Corneas from Lum⫺/⫺ and agematched Lum⫹/⫹ controls were used in these experiments at the postnatal stages indicated. Processing for transmission electron microscopy was as previously described (Birk and Trels-
2504 CHAKRAVARTI ET AL.
tad, 1984; Chakravarti et al., 2000). Brieﬂy, whole eyes were placed in ﬁxative and the corneas dissected. The corneas were ﬁxed in 4% paraformaldehyde/2.5% glutaraldehyde/ 0.1 M sodium cacodylate, pH 7.4, with 8.0 mM CaCl2 for a total of 2 h on ice, followed by post-ﬁxation with 1% osmium tetroxide for 1 hr, dehydrated to 50% ethanol, en bloc stained with ethanolic uranyl acetate and complete dehydration in a graded ethanol series, followed by propylene oxide. The corneas were inﬁltrated and embedded in a mixture of Embed 812, nadic methyl anhydride, dodecenylsuccinic anhydride, and DMP-30 (EM Sciences, Fort Washington, PA). Thick sections (1 m) were cut and stained with methylene blue-azur B for light microscopy and selection of speciﬁc regions for further analysis. Thin sections were prepared using a Reichert UCT ultramicrotome and a diamond knife. Staining was with 2% aqueous uranyl acetate followed by 1% phosphotungstic acid, pH 3.2. Sections were examined and photographed at 80 kV using a Hitachi 7000 transmission electron microscope. The microscope was calibrated using a line grating.
Fibril Measurements The central corneal stroma was divided into 4 equal regions. Analyses were done for both the anterior and posterior stroma. The anterior stroma was deﬁned as the region subjacent to the epithelium and the posterior stroma was adjacent to the endothelium. Both regions were photographed in the central portion. Micrographs were taken at 48,700⫻. Calibrated micrographs from each region were randomly chosen in a masked manner from the different regions. The micrographs were digitized and all diameters were measured within a 1.6-m2 mask. The mask was placed based on ﬁbril orientation, i.e., cross-section and absence of cells. Diameters were measured along the minor axis of ﬁbril cross-sections using a RM Biometrics-Bioquant Image Analysis System (Memphis, TN).
Statistical Methods The following statistical analyses of ﬁbril diameters were performed for
the anterior and posterior stroma. In the anterior stroma, there were a total of 59 negatives from 29 animals (5 animals per age/genotype combination, except for the 10-day Lum⫹/⫹ group with 4 animals, usually 2 negatives per animal, 1 ﬁeld per negative). In the posterior stroma, a total of 360 ﬁelds from 90 negatives from 28 animals (4 or 5 animals per age/genotype combination, usually 3 negatives per animal and always 4 ﬁelds per negative) were analyzed. The anterior and posterior ﬁbril diameters were measured in the same 28 animals. The cornea ﬁbril diameters in each measured ﬁeld in a negative were considered as a mixed distribution consisting of a dominant symmetric component contaminated with a relatively small percentage of outliers. To identify potential outliers in each measured ﬁeld, a well-established resistant outlier detection rule was utilized (Hoaglin et al., 1986). This rule labels as an outlier any observation that falls below Q1–1.5(Q3–Q1) or above Q3 ⫹ 1.5(Q3–Q1), where Q1 and Q3 are the ﬁrst and third sample quartiles, respectively. The ﬁrst and the third sample quartiles, Q1 and Q3, are the values where 25 and 75% of the ﬁbril diameters are less than or equal to Q1 and Q3, respectively (Rosner, 1995). Thus, the interval [Q1, Q3] contains the central 50% of the observations, and the sample inter-quartile range (Q3–Q1) is representative of the data spread. This rule is implemented in the usual box-plot data presentation. Also, extreme outliers were deﬁned as all observations below Q1–3(Q3–Q1) or above Q3 ⫹ 3(Q3–Q1) (Hoaglin et al., 1986). The numbers of large outliers (above Q3 ⫹ 1.5(Q3–Q1)) per ﬁeld were modeled in a generalized linear mixed effects model (Vonesh and Chinchilli, 1997), with the assumption of the negative binomial distribution, which provided a better ﬁt than the Poisson distribution (also often used to model count data). These models incorporated animal-to-animal and negativeto-negative variability, but without separating corresponding variance components. Two separate models were ﬁt to the anterior and posterior data. For the outlier-trimmed posterior cornea ﬁbril diameter data, the as-
sumption of a Gaussian distribution was reasonable. However, for the outlier-trimmed anterior cornea ﬁbril diameters, it was adequate to assume symmetric, but not Gaussian dominant component. Therefore, non-parametric measures (not dependent on any distribution assumptions and robust with respect to the presence of outliers) were used to summarize all of the outlier-trimmed data from each negative. We used the sample median as a measure of location and the sample inter-quartile range (Q3–Q1) as a measure of spread. The negative-speciﬁc medians and square roots of inter-quartile ranges were separately modeled in a linear mixed effects model incorporating animal-to-animal and negative-to-negative variability. In these models, the anterior and posterior outlier-trimmed data were combined and accounted for the correlation of measures from the same animals. Based on examination of residuals from these models, the model assumptions were adequate for these data.
ACKNOWLEDGMENTS We acknowledge the expert technical assistance of Biao Zuo and Diana W. Menezes. We thank Dr. Magnus Ho¨o¨k, Center for Extracellular Matrix Biology, Texas A&M University Health Science Center, Houston, for kindly providing the antibodies against osteoglycin. This work was supported by grants from the National Institutes of Health, EY05129 (D.E.B.) and EY11654 (S.C.).
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