Developmental Changes in Patterns of Expression of Tenascin-C Variants in the Human Cornea

Abstract

purpose. To study patterns of expression of alternatively spliced tenascin-C (TN-C) variants believed to mediate cellular activities in human corneal development.

methods. Serial sections of preterm, neonatal, child, and adult globes with normal anterior segments were labeled with monoclonal antibodies to TN-C. The antibodies included BC-4 and BC-8, which recognize epitopes in conserved domains of TN-C and can thus detect all TN-C variants, and BC-2, α-A2, α-A3, α-IIIB, TN11, and α-D, which bind to epitopes in alternatively spliced fibronectin type III repeats of TN-C. Bound antibodies were localized and visualized using an avidin-biotin complex–alkaline phosphatase technique.

results. BC-4 and BC-8 showed similar patterns of staining, widely observed in preterm corneas, less so in neonatal corneas, and restricted to the limbus in the child and adult. BC-2, α-A2, α-A3, α-IIIB, TN11, and α-D staining was largely localized in corneal epithelium (preterm and neonatal), limbal epithelium, mast cells, and matrix surrounding limbal vessels (preterm, neonatal, child, and adult).

conclusions. TN-C may play a role in corneal development and in growth and differentiation of stem cells because it is widely expressed in the preterm cornea, less so in the neonate, and is restricted to the limbus in the child and adult. The differential patterns of expression of TN-C variants in normal corneas (preterm and neonatal), and in the limbus (preterm, neonatal, child, and adult), suggest specific roles played by each variant, and cell type–specific expression of the different variants.

Tenascin-C (TN-C) is a matricellular protein ¹ believed to play important roles in tissue development, wound healing, and repair, because it mediates several cellular activities including cell adhesion and antiadhesion, migration, proliferation, and differentiation (reviewed by Chiquet–Eherismann et al. and Mackie ^{2 3} ). In humans, TN-C is encoded by a single gene localized within region q32-q34 of chromosome nine, ^{4 5} and thus the expression of a single protein would be expected. This is, however, not the case, because TN-C mRNA undergoes alternative splicing (Fig. 1) , generating variants that incorporate all (high M_r variants), some (intermediate M_r variants), or none (low M_r variants) of the fibronectin type III repeats, TNCfn-A1 to TNCfn-D. ^{6 7 8 9 10 11 12 13}  

The expression of alternatively spliced variants with different compositions of the TNCfn repeats, in part, explains the pleiotropic nature of TN-C. Functional studies have, to some extent, identified specific repeats that mediate cell antiadhesion (TNCfn-1 to -4 and TNCfn-A1 to -A4), migration (TNCfn-10 and TNCfn-11), and growth and differentiation (TNCfn-A2, TNCfn-A3, TNCfn-5, TNCfn-6, TNCfn-B, and TNCfn-D). ^{6 7 14 15 16 17 18 19 20 21 22 23}  

The expression of TN-C in the normal adult human cornea, in healing and repair, and in pathologic human corneas has been described in our previous studies ^{24 25 26} and by other researchers. ^{13 27 28 29 30 31} More recently, adult human corneas including normal corneas, those with wound healing and scarring, and corneas from cases of bullous keratopathy have been investigated for the presence of alternatively spliced TN-C mRNA ^{12 13} and the expression of TN-C variants. ^{13 25 26} However, there are currently few data regarding the patterns of expression of the TN-C variants during development of the human cornea. Therefore, it was envisaged that unraveling any differences in patterns of expression of TN-C variants in relation to human corneal development might enable a better understanding of the role of this glycoprotein in determining corneal morphology and development. 

Materials and Methods

Tissues

Specimens consisted of preterm, neonatal, child, and adult globes removed at autopsy or enucleated for retinoblastoma or choroidal melanoma, but without anterior segment disease. Histopathologic assessment of all globes indicated that all the corneas were within normal limits (see Table 1 for details). Immediately after removal, globes were fixed in 10% formalin, processed, and embedded in paraffin. Several serial sections (6 μm) encompassing the entire cornea (limbus to limbus) and scleral rim were obtained from each of the specimens for study. 

mAbs to Human TN-C

We used a panel of monoclonal antibodies (mAbs; Fig. 1 ), to human TN-C, designed to bind to epitopes localized in conserved domains of the TN-C molecule (BC-4 and BC-8) and alternatively spliced fibronectin type III repeats (BC-2, α-A2, α-A3, α-IIIB, TN11, andα -D). The former detect all TN-C variants, and the latter detect only those that incorporate the relevant alternatively spliced TNCfn repeats. ^{8 32 33}  

Immunohistochemical Staining Procedure

Sections were dewaxed in xylene, cleared in graded (99%, 80%, and 70%) industrial methylated spirit (IMS; BDH Laboratory Supplies, Poole, UK), and slides were immersed (5 minutes at room temperature[ RT]) in 0.05 M Tris-buffered saline (TBS, pH 7.5; Sigma–Aldrich, Poole, UK). The sections were then incubated (10 minutes at 37°C) with 1 mg/ml trypsin (Sigma–Aldrich) to expose masked epitopes. This was followed by immersion washes (two times for 2 minutes each at RT) of slides in TBS (pH 7.5). Subsequently, eight serial sections from each globe were each incubated (overnight at 4°C) with a selection of one of the eight mAbs to human TN-C described earlier. Slides were immersion washed (three times for 2 minutes each at RT) in TBS (pH 7.5), and the bound primary antibody was subsequently revealed using reagents of two substrate kits (Vectastain ABC-AP and Vector Red; Vector, Peterborough, UK). This entailed following procedures specified in the protocols of the two kits. In short, sections were incubated (30 minutes at RT) with 1:100 (vol:vol) biotinylated horse anti-mouse IgG1 to localize bound antibody. After immersion washing (three times for 2 minutes at RT) of the slides in TBS (pH 7.5), the localized bound antibody was visualized by incubating sections (30 minutes at RT) with avidin-biotin complex-alkaline phosphatase (ABC-AP). The slides were then immersion washed (three times at 2 minutes each at RT) in TBS (pH 7.5), and subsequently the sections were incubated (7 minutes at RT) with, a substrate of AP (Vector red). The substrate solution also contained levamisole to inhibit endogenous AP. Sections were rinsed (two times at 2 minutes each at RT) in distilled water, counterstained with Mayer’s hematoxylin (BDH), dehydrated in graded (70%, 80%, and 99%) IMS, cleared in xylene (four times at 3 minutes) and coverslipped. Negative IHC controls included substitution of mAbs to human TN-C with an irrelevant mAb, anti-desmin, clone D33, IgG1 (Dako, Cambridgeshire, UK), or normal horse serum (Vector). There was no staining seen in the negative controls. In addition to the entire cornea, all sections investigated in this study consisted of a scleral rim. The sclera provided a positive internal control for IHC detection of human TN-C. ^{24 25 26 27}  

In addition, as certain mAbs labeled what appeared to be mast cells, acid toluidine blue staining was used to confirm their phenotype. 

Microscopy and Photography

Using a light microscope (BH2; Olympus, Tokyo, Japan) equipped with a camera (C-35AD-4; Olympus), we examined serial sections from each of the specimens for IHC staining achieved with each of the mAbs. By exciting the substrate (Vector red) reaction product using a rhodamine filter system (535–560 nm), localized antibody reaction could be visualized as a fluorescent bright red color. Black and white micrographs were made (1600-PR film; Fuji, Tokyo, Japan) at a constant exposure time of 1.25 minutes. Observation was also made using transmitted light to reveal tissue and cellular morphology. For these, black and white micrographs were made with automatic exposure (TMY 400 film; Eastman Kodak, Rochester, NY). 

Results

Both mAbs detecting TN-C conserved domains showed similar patterns of staining. This showed the general pattern of expression of TN-C to be widespread in epithelial, stromal, and endothelial layers of the preterm corneas (Fig. 2A 2B ) but less so in the neonatal corneas where staining was absent from the stroma except adjacent to the corneoscleral junction (Fig. 2C) . In the child and adult, TN-C was detected only at the limbus (Fig. 2D) . 

Patterns of expression of specific TN-C variants in preterm, neonatal, child, and adult corneas (summarized in Table 2 ) were revealed by staining achieved with mAbs recognizing alternatively spliced TN-C variants. TN-C variants were not detected anywhere in the tissues where conserved domains could not be demonstrated. 

TN-C variants, including the alternatively spliced repeats A1 and/or A4, were detected in limbal and corneal epithelium (all preterm and neonates) and corneal endothelium of all preterm infants, but not in neonates (Table 2 ; Figs. 3A 3B ). In the children and adults, these variants were identified in the limbal epithelium only of child [C]-2 and all adults (Table 2 ; Fig. 3C ). 

TN-C variants containing repeat A2 were localized in the corneal epithelium (all preterm and neonates), stroma (preterm [P]-3 and neonate [N]-1), and corneal endothelium (all preterm and N-1; Table 2 ; Fig. 3D ). At the limbus, staining for the A2 repeat was localized to the epithelium (all preterm and neonates) and matrix around limbal vessels (P-3 and all neonates; Table 2 ). In the children and adults, staining for this repeat was restricted to the limbus where the epithelium (all children and adults [A]-1 and -2), matrix around the vessels and the corneal–scleral interface (C-2, A-1, and A-3) were labeled (Table 2) . 

TN-C variants containing the A3 repeat were detected in the corneal epithelium (P-3 and all neonates; Fig. 3E ) and corneal endothelium (P-3; Table 2 ). At the limbus, these variants were localized to the epithelium (P-1, P-2, and all neonates), matrix around vessels (N-1) and were associated with mast cells (all preterm and N-1; Table 2 ). In the children and adults, A3-containing TN-C variants were restricted to the limbus and were localized to the matrix around vessels and to mast cells (C-2, A-1, and A-3; Table 2 ). 

Repeat B-containing TN-C variants were noted in the corneal epithelium (all preterm and neonates), corneal stroma (P-2 and N-1) and corneal endothelium (P-1 and P-2; Table 2 ; Figs. 4A 4B ). At the limbus, these variants were localized to the epithelium, corneoscleral interface, and matrix around the vessels (all preterm and neonates) and were also associated with mast cells (P-3 and N-1; Table 2 ; Figs. 4A 4C ). In the children and adults, TN-C variants incorporating the B repeat were found only in the limbus, localized to the epithelium (all children, A-1, and A-2), matrix around vessels (all children and A-1) and the corneoscleral interface (C-1, A-2, and A-3). These variants were also associated with mast cells (all children; Table 2 ; Fig. 4D ). 

TN-C variants incorporating repeat C were seen in the corneal epithelium (all preterm, N-2, and N-3) and in corneal endothelium (P-1, P-2, N-2, and N-3; Table 2 ). At the limbus, these variants were localized to the epithelium and matrix around vessels (all preterm and neonates; Table 2 ; Figs. 4E 4F ). In the children and adults, TN-C variants with repeat C were restricted to the limbus where they were localized to the epithelium, matrix around vessels (all children and adults) and to mast cells (all children; Table 2 ; Figs. 4G 4H ). 

Repeat D-containing TN-C variants were detected in corneal epithelium and endothelium (P-3 and N-1; Table 2 ). At the limbus, these variants were localized to the epithelium (P-3 and all neonates) and to corneoscleral interface and matrix around vessels (N-1; Table 2 ; Figs. 4F 4G ). In the children and adults, TN-C variants incorporating the D repeat were restricted to the limbal epithelium (C-2 and A-1; Table 2 ). 

Discussion

In this study, we stained serial sections from developing and mature normal human corneas with eight mAbs (Fig. 1) specific to human TN-C. Two of these (BC-4 and BC-8) bind to epitopes in conserved domains ^{8 32} and thus revealed the general patterns of TN-C expression in these tissues (Fig. 2) . The others bind to epitopes localized to alternatively spliced fibronectin type III repeats (TNCfn-A1 to D) ^{8 32 33} and thus revealed the patterns of expression of TN-C variants (Table 2 ; Figs. 3 4 ). It was not possible to investigate the patterns of expression of TN-C variants incorporating repeats TNCfn-AD1 and TNCfn-AD2 because there were no relevant antibodies. Nevertheless, this is the first study to use a range of mAbs and serial sections to document the patterns of expression of TN-C variants in preterm, neonate, and child as well as in normal human adult corneas. 

Results (Table 2 ; Figs. 2 3 4 ) indicated that TN-C was abundantly expressed in normal preterm corneas, but less so in neonatal corneas, and was restricted to the limbus in the child and adult. These findings are consistent with the transient pattern of expression of TN-C which has been described in several developing tissues such as skin, lung, and tooth (reviewed by Chiquet–Eherismann et al. and Mackie), ^{2 3} and in agreement with a role for TN-C in corneal development previously suggested in the chicken. ^{34 35}  

Our results also showed that there are differential patterns of expression of TN-C variants. For example, only those containing repeats TNCfn-A2 and TNCfn-B were detected in the preterm and neonatal corneal stroma, whereas the epithelium and, to some extent, the endothelium of preterm and neonatal corneas were positive for several TN-C variants (Table 2 ; Figs. 3 4 ). At the limbus at all age groups examined, the epithelium, unlike the matrix and vessels, showed presence of a number of TN-C variants (Table 2 ; Figs. 3 4 ). 

The expression of TN-C, but not its variants, has been described in the epithelium of a 3-month-old fetal cornea, ²⁷ and in the limbus of adult corneas. ^{24 25 26 27 28 29 30 31} More recently, studies using reverse transcription–polymerase chain reaction (RT-PCR) have demonstrated the presence of TN-C mRNA isoforms in epithelium, stroma, and endothelium of normal adult corneas. ^{12 13} However, in agreement with our findings, TN-C proteins were not detected in either of these studies. There is, as yet, no explanation for the absence of expression of TN-C proteins in normal child and adult human corneas. ^{24 25 26 27 28 29 30 31} There is (other than at the limbus) interspecies variation in that the expression in the mouse ³⁶ is similar to that in humans, whereas in the rabbit, TN-C has been immunodetected in the entire epithelium of normal adult rabbit corneas. ^{37 38 39} The differential patterns of localization in the human cornea may be important in corneal development and in remodeling by mediating dynamic cellular functions such as migration, proliferation, and differentiation. A 220-kDa TN-C variant was found to be associated with migrating epithelium of developing avian cornea ³⁴ although the authors unfortunately did not define the composition of the alternatively spliced repeats. Other investigations have shown that repeats A2, A3, B, and D can mediate neurite outgrowth and neuronal differentiation ^{16 19 20 21 22 23} whereas variants incorporating repeats A2, A3, and B promote cell migration in vitro and are expressed by migrating cells such as glia and osteoblasts. ^{21 22 23}  

A novel finding of retention of TN-C variants incorporating repeat TNCfn-C in mast cells in the child and adult corneas studied is probably related to the presence of vascularization at the limbus, for mast cells are known to be associated with vessel formation. ^{23 40} This may provide a favorable matrix for mast cell–associated angiogenesis and cell proliferation as noted by Carnemolla et al. ³³  

Retained expression of TN-C in the child and adult normal limbus (Table 2 ; Fig. 2D ) is of interest. TN-C expression is seen in normal, continuously renewing adult tissues such as the epithelial–mesenchymal interface of the gastrointestinal tract ² and is transiently expressed in the placenta and endometrium—tissues known to exhibit dynamic cellular activity. ^{41 42} The limbus is a region of high cellular activity involving proliferation and differentiation of stem cells. ⁴³ Thus, TN-C variants in the limbus may provide a favorable milieu for continued corneal epithelial replenishment and vascularization. The various TN-C variants found in the limbus of these corneas may be due to cell-cycle–dependent TN-C mRNA alternative splicing. ⁴⁴  

In summary, this study has shown that in the normal human cornea TN-C is widely expressed in the preterm infant, less so in the neonate, and is restricted to the limbus in the child and adult. This pattern of expression supports the view that TN-C plays a role in corneal development. Retained expression of TN-C in the limbus of normal child and adult corneas may indicate a role in the growth and differentiation of limbal stem cells and the continuous replenishment of corneal epithelium. Furthermore, TN-C variants are differentially expressed in the preterm and neonatal corneal layers, and in the limbus of preterm, neonatal, child and adult corneas. This may indicate specific roles played by each variant and, possibly, cell type–specific expression of the different variants at different times during development and remodeling. 

 Supported by the Manchester Royal Eye Hospital Endowments.

 Submitted for publication September 28, 1999; revised February 29 and July 13, 2000; accepted July 28, 2000.

 Commercial relationships policy: N.

 

Figure 1.

 

One of the six identical TN-C monomers, its modular domains, and location of epitopes for the various mAbs. As a result of alternative splicing of TN-C mRNA in the region encoding repeats TNCfn-A1 to TNCfn-D, monomers may vary in size from approximately 190 to 320 kDa. ^{7 9 10 11} Epitopes to which the various mAbs bind are indicated by a, BC-4; b, BC-8; c, BC-2; d, α-A2; e, α-A3; f,α -IIIB; g, TN11; and h, α-D. ^{8 32 33}

Table 1.

 

Normal Corneas: Demographic, Clinical, and Pathologic Details

Cornea	Clinical and Pathologic Details
P-1 and P-2	Eyes obtained after death from a premature boy (24 weeks gestation). Early retinopathy of prematurity. Died 1 day later from bronchopneumonia. Normal corneas and anterior segment.
P-3	Eye from a stillborn girl (34 weeks gestation). Early retinoblastoma. Normal cornea and anterior segment.
N-1	Eye from a male neonate (5 weeks postpartum). Nonaccidental death. Normal cornea and anterior segment.
N-2 and N-3	Eyes from a male neonate (5 months postpartum), who was premature (27 weeks gestation) birth. On intensive care (continuous ventilatory oxygen). Died of bronchopulmonary dysplasia. Minimal retinopathy of prematurity. Normal corneas and anterior segment.
C-1	Eye from a male child (2 year). Enucleated for retinoblastoma. Normal cornea and anterior segment.
C-2	Eye from a male child (2 year). Enucleated for retinoblastoma. Normal cornea and anterior segment.
A-1	Eye from a 40-year-old man. Enucleated for malignant choroidal melanoma. Normal cornea and anterior segment.
A-2	Eye from 62-year-old man. Enucleated for malignant choroidal melanoma. Normal cornea and anterior segment.
A-3	Eye from a 90-year-old man. Enucleated for malignant choroidal melanoma. Normal cornea and anterior segment.

Figure 2.

 

General patterns of TN-C expression in developing and mature normal human corneas detected using pan-TN-C mAbs. (A) Peripheral cornea and limbus. (B) Central cornea. TN-C was present in the entire preterm corneal epithelium, stroma, endothelium, and corneal–scleral interface. (C) Peripheral cornea and limbus. TN-C was restricted to the limbus at the scleral spur and corneoscleral interface in child (not shown) and adult (D) corneas. However, in the neonate (C), in contrast to the child and adult, it was present in the limbal epithelium and peripheral corneal stroma as well. Montages; magnifications, ×370.

Table 2.

 

The Expression of TN-C Variants in the Human Cornea

	A1 and/or A4	A2	A3	B	C	D
Preterm
Limbus
Epithelium	+	+	−	+	+	+
Vessels	−	+	−	+	+	−
Corneal–scleral interface	−	−	−	+	−	−
Cornea
Epithelium	+	+	+	+	+	+
Stroma	−	+	−	+	−	−
Endothelium	+	+	+	+	+	+
Neonate
Limbus
Epithelium	+	+	−	+	+	+
Vessels	−	+	+	+	+	+
Corneal–scleral interface	−	−	−	+	−	−
Cornea
Epithelium	+	+	+	+	+	+
Stroma	−	+	−	+	−	−
Endothelium	−	+	−	−	+	+
Child/adult
Limbus
Epithelium	+	+	−	+	+	+
Vessels	−	+	+	+	+	−
Corneal–scleral interface	−	+	−	+	−	+
Cornea
Epithelium	−	−	−	−	−	−
Stroma	−	−	−	−	−	−
Endothelium	−	−	−	−	−	−

 A1, A2, A3, A4, B, C, and D refer to alternatively spliced fibronectin type III repeats of TN-C. The presence or absence of TN-C variant(s) incorporating the specified repeat is denoted by + or− , respectively. Because mAb BC-2 binds to epitopes found in two repeats (A1 and A4), it was assumed that positive staining indicated the expression of TN-C variant(s) with any or both of these repeats.

Figure 3.

 

Localization of TN-C variants in developing and mature normal human corneas. (A, B) Central cornea. TN-C variants incorporating repeats A1 and/or A4 were seen in basal epithelium (arrowheads) of preterm and neonatal corneas. (C) Limbus. In the child and adult (not shown), repeat A1- and/or A4-containing variants were restricted to the limbal epithelium (arrows). (D) Central cornea. In preterm cornea, variants incorporating the A2 repeat were seen in epithelium (epi) and stroma (stro). (E) Central cornea. Variants containing the A3 repeat were localized in basal epithelium (arrowheads) and stroma (stro) of neonatal cornea. (F) Peripheral cornea and limbus. Variants with repeat D were localized in the entire limbal epithelium (arrows) and limbal matrix (lm) of the preterm cornea. (G) Peripheral cornea and limbus. In the neonate repeat D-containing variants were present mainly in limbal basal epithelial cells (arrows) and to a lesser extent in the limbal matrix (lm). (H) Peripheral cornea and limbus. Preterm negative control. Magnification: (A through E), ×370; (F, G, and H),× 185.

Figure 4.

 

Localization of TN-C variants in developing and mature normal human corneas. (A) Peripheral cornea and limbus. (B) Central cornea. Repeat B–containing TN-C variants were seen in the limbus (arrows), around vessels (v), and in corneal epithelium (epi) of preterm cornea. (C) Peripheral cornea and limbus. In the neonate these variants were heavily localized in the limbal epithelium (arrows) and matrix (lm), especially around vessels (v), diminishing toward the corneal axis (arrowhead). (D) Peripheral cornea and limbus. In the adult these variants were restricted to the limbus, notably in the limbal matrix (lm), around some vessels (v), and, to a lesser extent, in the superficial layer of limbal epithelium (arrows). (E) Central cornea. In preterm repeat C–containing variants were present in the entire epithelium (arrowheads). (F) Central cornea. In neonate repeat C–containing variants were seen in occasional epithelial cells (arrowheads). (G, H) Limbus. In the child and adult (not shown), these variants were restricted to the limbal epithelium (arrow), matrix around limbal vessels (v), and some cells within the limbus. At a higher magnification of the same cornea, some of these cells are identified as mast cells by acid toluidine blue staining (H, inset). Magnifications: (A, B, E and F), ×370; (C), ×185; (D, G), ×450.

The authors thank Luciano Zardi and coworkers at Centro Biotecnologie Avanzate, Genova, Italy, for the kind gift of monoclonal antibodies to human TN-C.