Rabbit Conjunctival and Corneal Epithelial Cells Belong to Two Separate Lineages

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1 Rabbit Conjunctival and Corneal Epithelial Cells Belong to Two Separate Lineages Zhi-Gang Wei,* Tung-Tien Sun,-\ and Robert M. Lavker* Purpose. This study investigated rabbit conjunctival and corneal epithelial cells to determine if they belong to two separate lineages. Methods. Rabbit corneal, limbal, and conjunctival epithelial cells were isolated and grown in Dulbecco's minimum essential media and 20% fetal bovine serum in the presence of mitomycin-treated 3T3 feeder cells. After reaching 80% confluence, 3T3 feeder cells and any contaminating fibroblasts were removed, and epithelial cells were resuspended in fresh Dulbecco's minimum essential media. Aliquots containing 5 X 10 h cells were placed subcutaneously into the flanks of athymic mice, which subsequently formed small nodules. At 2, 4, 6, 8, 14, 21, and 28 days, athymic mice were killed and the nodules (epithelial cyst) were excised for light and transmission electron microscopic examination and histochemical and cell kinetic analyses. Results. Within 2 days after injection of single-cell suspensions, cells aggregated to form cysts lined with a stratified squamous epithelium, the structure of which resembled the original in vivo donor sites by 8 days. Limbal- and corneal-derived cysts were comprised only of glycogen-rich stratified epithelial cells. In contrast, only cysts arising from cultured conjunctival cells contained periodic acid-schiff-positive cells with a goblet cell structure interspersed among stratified epithelial cells. Furthermore, cystic epithelium of conjunctival origin did not accumulate glycogen. Conclusions. To determine whether distinct phenotypes are caused by intrinsic divergence or by environmental modulation, the behavior of cells can be monitored in an identical in vivo growth environment. The athymic mouse provides such a permissive growth environment for cultured corneal, limbal, and conjunctival epithelial cells. All these cells reproduced their in vivo phenotype when placed in the athymic mouse. Thus, these findings provide the strongest evidence to date that the corneal-limbal lineage is distinct from the conjunctival lineage. These data also support the idea that the progenitor of goblet cells does not reside in the corneal-limbal epithelial compartment. Invest Ophthalmol Vis Sci. 1996; 37: A he anterior surface of the eye is covered by contiguous but histologically distinguishable stratified squamous epithelia that overlie the cornea, limbus, and conjunctiva. The mechanisms by which these varied ocular epithelial phenotypes are established and maintained are unknown, but at least two hypotheses have From the * Department of Dermatology, University of Pennsylvania School of Medicine, Philadelphia, and the f Epithelial Biology Unit, Ronald O. Perelman Department of Dermatology and Department of Pharmacology, New York University Medical Center, Kaplan Comprehensive Cancer Center, New York. Supported by National Institutes of Health grants EY06769 and AR07465 (RML) and EY04722 (ITS). Submitted for publication July 3, 1995; revised November 14, 1995; accepted November 16, froprietary interest category: N. Reprint requests: Robert M. lmvker, Department of Dermatology, University of Pennsylvania School of Medicine, Clinical Research Building/235 A, 415 Curie Boulevard, Philadelphia, PA been proposed: Distinct epithelial cell types give rise to the various regions, and the stroma dictates the pattern of epithelial differentiation. That stromal factors can, in fact, influence epithelial differentiation is supported by studies of the epithelial phenotype expressed when conjunctival epithelium migrates over the corneal stroma after "total" corneal epithelial loss. Specifically, in the presence of vascularization of the corneal stroma, 1 " 5 a conjunctival-like phenotype (conjunctivalization) will persist in the epithelium that covers the corneal stroma. However, if blood vessels are not present in the corneal stroma, the conjunctival epithelium assumes a corneal-like phenotype, as shown using the light microscope. These studies clearly indicate that stromal factors can influence epithelial phenotype; however, they do not negate the Investigative Ophthalmology & Visual Science, March 1996, Vol. 37, No. 4 Copyright Association for Research in Vision and Ophthalmology 523

2 524 Investigative Ophthalmology & Visual Science, March 1996, Vol. 37, No. 4 possibility that different epithelial stem cells are important determinants of the distinct ocular epithelial phenotypes. The relative contributions of distinct cell lineage (that is, intrinsic divergence) compared with external modulation can be determined by monitoring the behavior of epithelial cells when they are placed in identical growth environments. If two epithelia behave differently even under identical conditions, the epithelia are probably intrinsically different. For example, previously we showed that when single-cell suspensions of cultured rabbit esophageal, corneal, and epidermal keratinocytes are injected subcutaneously into athymic mice, the cells regain their distinct in vivo morphologic and biochemical phenotypes; that is, the corneal, epidermal, and esophageal keratinocytes formed epithelia that were nonkeratinized, keratinized, and parakeratinized, respectively. 6 7 These data show that although external signals can modulate to a significant degree the differentiation programs of keratinocytes, the three cell types are intrinsically divergent. In the present study, we analyzed the in vivo differentiation of cultured corneal, limbal, and conjunctival epithelial cells. This in vivo model permits the cells to reproduce their original tissue phenotype faithfully. Specifically, in cysts arising from cultured conjunctival epithelial cells, periodic acid-schiff-positive cells with a goblet cell structure were interspersed with stratified columnar epithelial cells. In contrast, limbal- and corneal-derived cysts contained only stratified squamous epithelial cells. This clearly supports the hypothesis that corneal and limbal keratinocytes are intrinsically different from conjunctival keratinocytes, and it provides strong evidence that the progenitor of goblet cells does not reside in the corneal-limbal epithelial compartment. MATERIALS AND METHODS All experimental procedures conformed to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the University of Pennsylvania Animal Care and Ethics Committee. Isolation and Cultivation of Rabbit Corneal, Limbal, and Conjunctival Cells Female New Zealand white rabbits weighing 3 to 4 kg were killed by intravenous injection of T-61 euthanasia solution. Keratinocytes from the various portions of the anterior segment were isolated as previously described. 8 Briefly, the central corneal button (5-mm diameter), a 2-mm-wide limbal tissue, and the whole sheet of conjunctival tissue were removed with scissors. In those experiments in which specific zones of conjunctival epithelium were studied, after trimming the whole sheet of conjunctival tissue off the Tenon tissue, a specific zone of the conjunctiva (bulbar, fornix, or palpebral) was conformed to the profile of a sterile paraffin sheet with a central depression diameter of approximately 4 mm. The specific conjunctival regions and the corneal and limbal epithelium were treated with Dispase II (Boehringer Mannheim, Indianapolis, IN) at 37 C for 3 hours under 5% CO 2 and 95% air. The detached epithelium was isolated by light scraping and was dissociated into single cells and small clumps by pipetting. The cells were then plated at a concentration of 2 X 10 5 per 100-mm dish in Dulbecco's minimum essential medium containing 20% fetal bovine serum in the presence of mitomycin C-treated 3T3 feeder cells, with medium changed twice each week. Implantation of Cultured Corneal, Limbal, and Conjunctival Keratinocytes Into Athymic Mice After reaching 80% to 90% cell confluence, the 3T3 feeder cells and any contaminating rabbit fibroblasts were removed selectively by treatment with ethylenediaminetetraacetic acid. Epithelial cells were detached with light trypsinization (0.125% trypsin in 0.01% ethylenediaminetetraacetic acid at 37 C for 5 minutes) and resuspended in fresh Dulbecco's minimum essential media. Aliquots (0.5 ml) containing 5 X 10 6 cells were injected with an 18-gauge needle subcutaneously into the flanks of athymic mice. The soft swelling produced at the site of injection usually decreased in size within 24 to 48 hours, presumably because of the media's absorption. A small firm nodule subsequently formed during the ensuing 2 to. 3 weeks, reaching a maximum diameter of 3 to 5 mm, and then began to decrease slowly. Animals were killed by cervical dislocation at 2, 4, 6, 8,14, 21, and 28 days, and the nodules were excised for light and transmission electron microscopic examinations and cell kinetic, histochemical, and immunocytochemical analyses. Tritiated Thymidine Incorporation To measure the proliferative rate of the various epithelial cysts, tritiated thymidine was used to detect cells in S phase. Two hours before they were killed, athymic mice received intraperitoneal injections of 150 //Ci tritiated thymidine (specific activity, 82.7 Ci/mmol; New England Nuclear, Boston, MA) in 0.15 ml sterile phosphate-buffered saline. After the mice were killed, nodules were removed, fixed, and processed for autoradiographic examination as previously described. 9 Histologic and Electron Microscopic Analyses Nodules used for cell kinetic and light microscopic analyses were fixed in 10% formalin in phosphatebuffered saline, embedded in JB-4 plastic embedding

3 Modulation of Corneal, Iimbal, and Conjunctival Keratinocytes 525 medium, cut at 3 mm, and stained with either Alcian blue-periodic acid-schiff and hematoxylin or hematoxylin and eosin. Nodules used for transmission electron microscopic analysis were processed as described. 7 RESULTS In Vivo Compared With In Vitro Differentiation The corneal-iimbal epithelia consist of five to six distinct cell layers that are bound tightly with minimal intercellular space (Figs, la, b). In contrast, conjunctival epithelium consists of only two or three layers of columnar-shaped cells (Figs, la, c-f) that appear loosely packed because they are separated by wide intercellular spaces, with many goblet cells interspersed (Figs, la, c-f). Colonies formed from corneal, Iimbal, and all the conjunctival epithelia were morphologically similar, forming four to six cell layers. Ultrastructurally, cultured corneal and Iimbal keratinocytes had relatively straight cell-cell boarders, with few intercellular spaces (Fig. 2a), whereas conjunctival epithelial cells had many intercellular microvillus cell extensions with relatively large intercellular space (Fig. 2b). Cells containing mucous granules typical of those seen in goblet cells were not observed in these conjunctival colonies (Fig. 2b). General Characteristics of Ocular Epithelial Cysts When a single-cell suspension of cultured keratinocytes (5 X 10 b cells) was injected subcutaneously into the trunk of athymic mice, the cells form epithelial cysts. Within 2 days, the implanted corneal, Iimbal, and conjunctival cells were similarly organized into cysts composed of a contiguous stratified epithelium with clearly delineated inner and outer boundaries (Fig. 3a). All cysts were bounded by a mouse stroma that consisted of densely packed, elongated cells (presumably fibroblasts) and an extensive network of small and large vessels with prominent endothelial cells (Figs. 3a, c). When tritiated thymidine was administered as a single pulse, labeled nuclei were observed primarily in the basal cell layer, although labeled cells were also observed in a suprabasal position (Fig. 3b). With time, the walls of the cysts became thinner, and by 21 days many cysts began to show signs of degeneration. Cysts of Limbal and Corneal Epithelia By 7 days, the structure of the cystic limbal and corneal epithelia appeared similar to their in vivo counterparts (Fig. 4a). Limbal and corneal cells organized into a stratified squamous epithelium that consisted of a layer of basal cells, two to four layers of wing cells, and one or two layers of superficial cells (Fig. 4a). Epithelial cells were strongly stained with periodic acid-schiff (Fig. 4b); this staining was due to glycogen because it could be abolished by diastase pretreatment (Fig. 4c). 10 At no time were any goblet cells detected within these limbal- and corneal-derived epithelia (Figs. 4a-c, 5a-c). With time, differences were noted between the corneal- and limbal-derived cysts. Comparison of 21-day-old limbal (Fig. 6a) and corneal cysts (Fig. 6b) revealed that corneal cysts appeared more disorganized, consisting of a heterogeneously sized basal cell population, and focal areas of concentrically arranged cells were interspersed in regions where cells remained stratified (Fig. 6b). Conversely, limhal epithelial cells yielded nodules containing well-formed viable epithelial cysts composed of small, round-tocuboidal basal cells, three to four layers of wing cells, and one or two layers of flattened superficial cells (Fig. 6a). Tritiated-thymidine-labeled nuclei were noted in basal and suprabasal cells of both corneal- and limbalderived epithelial cysts; however, labeled nuclei were observed more frequently in the limbal-derived epithelia. Overall, the 21-day-old, limbal-derived epithelial cysts appeared more viable than the corneal-derived epithelial cysts. Ultrastructural examination revealed cystic epithelia that closely resembled the in vivo corneal and limbal epithelia in almost every detail (Fig. 5a), including the formation of a fully formed basement membrane, closely opposed cell-to-cell interfaces, the accumulation of rosettes of electron-dense granules characteristic of glycogen (Fig. 5b)," and small membrane-bound vesicles in the superficial cells (Fig. 5c) Cysts of Conjunctival Epithelium The cultured conjunctival cells organized into a stratified columnar epithelium consisting of two to three layers of nonkeratinizing squamous epithelium containing many Alcian Blue- and periodic acid-schiff-positive cells with a goblet cell structure (Fig. 4d). This stain was due to mucin, instead of glycogen, because it was not diminished by pretreatment with diastase (Figs. 4e, f). Cysts derived from fornical epithelium (Figs. 7c, d) consisted of a single layer of cuboidal basal cells, three to four layers of columnar epithelial cells, and many fully formed goblet cells (Figs. 7c, d). In contrast, bulbar- and palpebral-derived cysts (Figs. 7a, b, e) were thinner, appeared more squamous due to the presence of flattened epithelial cells (Figs. 7a, b, e), and contained relatively fewer goblet cells. Ultrastructural examination revealed conjunctival epithelial cells interspersed with goblet cells, both of which were in contact with a well-developed basement membrane (Fig. 5d). Like their in vivo counterparts, the epithelial cells had markedly infolded and interdigitating

4 526 Investigative Ophthalmology & Visual Science, March 1996, Vol. 37, No um FIGURE 1. Morphologic definition of rabbit corneal, limbal, and conjunctival epithelia. (a) Survey light micrograph showing the relationship of the corneal (C) and limbal (L) epithelia to the bulbar (B), fornical (F), and paipebral (P) zones of the conjunctiva, (b) Light micrograph of a portion of the limbal epithelium showing an undulated epithelium resting on a highly vascularized stroma. (c) The bulbar conjunctival epithelium is dramatically thinner than the limbal epithelium, and epithelial cells are more columnar. In addition, some goblet cells (G) are present among the epithelial cells, (d) As the fornical zone is approached, the epithelium increases to three or four cell layers, and goblet cells also become more numerous, (e) The fornical epithelium is considerably thicker than the bulbar epithelium and contains many goblet cells that exist as single cells, (f) A portion of the paipebral epithelium near the fornical zone characterized by a diminution of goblet cells and abundant lymphoid tissue, (g) The paipebral epithelium closer to the lid margin is considerably different because it becomes more stratified and goblet cells are absent.

5 Modulation of Cornea], Linibal, and Conjunctival Keratinocytes 527 FIGURE 2. Ultrastructure of cultured rabbit (a) corneal and (b) fornical conjunctival epidielial cells. Keratinocytes were isolated from respective tissues using Dispase and plated in Dulbecco's minimum essential medium containing 20% fetal bovine serum in the presence of mitomycin C-treated 3T3 feeder cells. Comeal epithelial cells are filled with ribosomes (R), mitochondria (M), keratin filaments (K), and typical desmosomal junctions (D) between the closely apposed stratified cells. Many intercellular microvillus cell extensions (arrows) and wide intracellular space are the major diiferences noted in the cultured conjunctival epithelial cells (b). Fornical conjunctival epithelial cells showed no evidence of goblet cell differentiation as defined by the formation of membrane-bound granules filled with flocculent material. membranes and relatively wide intercellular spaces. The distal membranes of the outermost epithelial cells were organized into a series of microvilli. Mucous granule internal structure consisted of a mixture of fine granular and flocculent material, producing an electron-lucent appearance (Fig. 5e). The individual mucin granules were observed in close contact with profiles of the rough endoplasmic reticulum (Fig. 5e). The center of the goblet cell was occupied by a mass of tightly packed mucin granules, producing the characteristic wine goblet appearance (Figs. 5d). In the apical portion of the goblet cell, some mucous granules were separated by small strands of cytoplasm, and other granules appeared to be coalesced, as judged by a loss of the limiting membranes between adjacent granules (Fig. 5f). Goblet cells within the cyst displayed a surface consisting of microvilli when in a nonsecretory phase (Fig. 5f), or a surface composed of coalesced mucous droplets with breaks or ruptures, protruding mucous granules, and "free mucous" appearing in the lumen (Fig. 5g). DISCUSSION Corneal-Limbal Epithelium and Conjunctival Epithelium: Two Distinct Cell Lineages We have shown that cultured corneal, limbal, and conjunctival epithelial cells, when placed subcutaneously in athymic mice, faithfully reproduce dieir in vivo phenotype. Thus morphologic analysis revealed that cysts derived from corneal and limbal epithelial cells consist of a stratified squamous epithelium devoid of goblet cells (Figs. 4a c), whereas epithelial cells from all conjunctival zones yield cysts composed of stratified columnar cells interspersed with goblet cells (Figs. 4de). Furthermore, the epithelium of the corneal-derived cysts contain material that stains positively with periodic acid-schiff, is sensitive to diastase, and is organized into clusters or rosettes of electron-dense granules similar to glycogen particles. This is consistent with the large glycogen stores that are a characteristic feature of corneal epithelium in vivo and may serve as a primary energy source In contrast, glycogen is not detected in the epithelia of the conjunctivalderived cysts, consistent with the' in vivo phenotype of these cells. Because the two distinct epithelial.phenotypes are faithfully reproduced in the context of a common stroma, stromal influences probably are not a major factor in determining the phenotypes of anterior ocular epithelia. Rather a more likely explanation for the morphologic and biochemical differences that we observed among the anterior ocular epithelia is that these cells are derived from distinct precursors. Sup-

6 528 Investigative Ophthalmology 8c Visual Science, March 1996, Vol. 37, No. 4 4c 4f 10 H m porting this possibility are studies 8 on the keratin patterns of cultured corneal, limbal, and conjunctival keratinocytes. Previously we showed that cultured corneal and limbal epithelial cells synthesize an identical set of keratins, including large quantities of the K3 and K12 markers of corneal-type differentiation, whereas all three types of conjunctival epithelial cells display another keratin pattern, with large amounts of simple epithelial keratins, but only traces of K3/K12 keratins. 8 In these studies, all cells were maintained under identical growth conditions. Thus the observed variation in keratin pattern strongly suggests that conjunctival epithelium represents a committed cell population that is intrinsically distinct from the corneal limbal epithelial lineage. 8 The most straightforward interpretation of our current data is that the corneal-limbal and the conjunctival compartments are governed by two distinct populations of stem cells. Our finding that corneal-limbal epithelia and conjunctival epithelia are not equipotent may be relevant to the issue of conjunctival transdifferentiation. In this process, conjunctival epithelium assumes some, but not all, of the characteristics of corneal epithelium when it migrates to the corneal stroma ~ 18 For ex-

7 Modulation of Cornea!, Limbal, and Conjunctiva! Keratinocytes 529 ngure 3. Morphologic features of an early epithelial cyst, (a) Survey light micrograph showing the relationship of an epithelial cyst formed 2 days after subcutaneous injection of cultured rabbit conjunctival epithelial cells into athymic mice. Similar morphologic features of the cyst were noted at the early time points (2 to 6 days) when cultured rabbit corneal and limbal keratinocytes were subcutaneously injected. Cultured keratinocytes are organized into a stratified epithelium (E) bounded by mouse stroma (S) and a lumen filled with cell debris (CD). Epidermis, Ep; hair follicles, F; panniculus carnosa, Pc. (b) The epithelium consists of small cuboidal basal cells (B), many of which have labeled nuclei (arrows) after a single pulse administration of tritiated thymidine. In the innermost portion of the viable epithelium, cells are larger and more stratified. CD, cell debris, (c) A portion of the highly vascularized (V) mouse stroma adjacent to the cyst consisting of densely packed elongated fibroblasts (F) and mononuclear cells (*). FIGURE 4. Comparison of epithelial cysts 21 days after subcutaneous injection of cultured rabbit corneal and conjunctival epithelial cells, (a) Stratified squamous epithelium from corneal-derived cells consists of a single layer of basal cells (B), two to three layers of wing cells (W), and one or two layers of superficial cells (S). Proliferation of basal and suprabasal cells is detected by tritiated thymidine incorporation (arrows). (b) A corneal-derived epithelial cyst stained with Alcian Blue and periodic acid-schiff (PAS) that shows many red-staining granules throughout the epithelial cells, (c) Diastase pretreatment of sections results in a marked reduction in PAS staining material indicative of glycogen. (d) Stratified columnar epithelium from conjunctiva-derived cysts consists of several layers of columnar epithelial cells interspersed with Alcian Blue- and PAS-stained goblet cells (arrows). Proliferation of basal and suprabasal cells is detected by tritiated thymidine incorporation (arrowheads), (e) Conjunctiva-derived epithelial cyst stained with Alcian Blue and PAS showing many magenta-staining goblet cells, (f) Pretreatment of sections with Diastase before staining with Alcian Blue and PAS had no effect on goblet cell staining, indicating the presence of mucin. ample, whereas the conjunctival-derived corneal epithelium appears normal when viewed with the light microscope, electron microscopic examination reveals the presence of immature goblet cells and widened intercellular spaces features that are typical of conjunctival epithelium. 19 In addition, the glycogen content and several other biochemical properties of conjunctival-derived corneal epithelium remain abnormal long after the transdifferentiation process is completed. 15 And, finally, in the presence of corneal stromal, the conjunctival-derived corneal epithelial phenotype can be modulated by the presence of vascularization, 2 photothrombotic occlusion of preexisting vascularization, 20 and changes in supplies of vitamin A to the corneas Thus although the stroma clearly influences ocular epithelial phenotype, it cannot induce a complete and irreversible transdifferentiation of conjunctival epithelium to corneal epithelium. Our data suggest a logical explanation for this finding: The two epithelia arise from distinct cell types. Maintenance of the Physical Boundary That the limbal-corneal epithelia and (bulbar) conjunctival epithelia clearly belong to two distinct cell lineages raises this question: What maintains their physical boundaries in vivo? More specifically, what prevents the conjunctival cells from invading into the corneal-limbal epithelia proper, and vice versa? This question is of general importance because a similar mechanism may operate to maintain the boundaries of neighboring cell populations in many organs. Examples of these include the junctions of various oral mucosal epithelia, the lip and facial epidermis, the esophageal and stomach epithelia, and the rectal epithelium and epidermis. Perturbation of the correct cell boundaries in any of these cases can cause major dysfunction of the involved organ. Although in the past little attention was paid to this subject, existing data suggest that basement membrane heterogeneity may play a role in forming and maintaining these boundaries. First, many cells exhibit remarkable specificity in their binding and attachment to different isotypes of one molecule of extracellular matrix. For example, human foreskin keratinocytes bind kalinin, a laminin isotype (laminin 5) and a component of anchoring fibrils, much better than they do the lamininnidogen complex. 23 Second, recent evidence revealed remarkable heterogeneity in the basement membrane, not only among those of different epithelia but even within a given epithelium. For example, Khosla et al 24 recently demonstrated heterogeneity in the distribution of glycosaminoglycan reactivity in basement membrane microdomains between the different levels of pulmonary airway epithelium. Thus, cell-binding specificity in combination with basement membrane heterogeneity can result in differential cell-substra-

8 530 Investigative Ophthalmology & Visual Science, March 1996, Vol. 37, No. 4 FIGURE 5. Comparison of the ultrastructure of corneal-derived (a) and conjunctival-derived (b) epithelial cysts, (a) Epithelium from a corneal-derived cyst is composed of basal (B), wing (W), and superficial (S) cells. Adjacent cells are closely apposed with many desmosomal junctions (D). (b) Portion of the epithelial cell cytoplasm showing many keratin filaments (K) and clusters or rosettes of electron-dense particles (arrows) typical of glycogen. (c) Portion of cytoplasm from a superficial cell containing filaments and small membranebound vesicles (V). (d) Epithelium from a conjunctiva-derived cyst showing goblet cells (G) interspersed with epithelial (E) cells. In contrast to the corneal-derived epithelium, conjunctivai epithelial cells display prominent microvillus extensions and wide intercellular spaces {arrows). A mass of tightly packed mucus granules (M)fillsmost of the goblet cell, (e) Individual mucin granules (M) are in close contact with profiles of the rough endoplasmic reticulum (*). (f) The apical portion of a nonsecretory goblet cell consists of a mat of fine filaments (F) that separate the mucus granules (M). The surface is organized into microvillus extensions (MV). (g) The apical portion of a goblet cell depicting coalesced mucus granules (M) in the process of being extruded or secreted (arrows) into the lumen (L).

9 Modulation of Corneal, Limbal, and Conjunctiva! Keratinocytes 531 FIGURE 6. Morphologic features of mature limbal- and corneal-derived epithelial cysts. A 21- day-old limbal-derived (a) epithelial cyst contains basal cells with many tritiated thymidinelabeled nuclei (arrows) compared widi a similarly aged corneal-derived (b) cyst. A corneaderived cyst (b) contains areas where cells are arranged in nests (*), yielding a more disorganized appearance, whereas the limbal-derived cyst (a) still maintains a more orderly, stratified squamous organization. FIGURE 7. Comparison of epidielial cysts derived from different zones of the conjunctiva. Epithelium formed 8 days after subcutaneous injection of cultured rabbit (a) bulbar and (b) palpebral conjunctival cells consists of two to four layers of epithelial cells with an occasional goblet cell (G). (c) In contrast, the epithelium formed 8 days after subcutaneous injection of cultured rabbit fornical cells is considerably thicker and contains many goblet cells (G). (d) The epithelium formed 21 days after subcutaneous injection of fornical cells consists of four to six layers of epithelial cells interspersed with goblet cells (G). (e) The epithelium from similarly aged cysts derived from palpebral epithelial cells is significantly thinner, more disorganized, and contains few goblet cells. Basal cells with tritiated diymidine-labeled nuclei (arrows) can still be observed in epithelium from 21-day-old fornicalderived cysts (d) but are rarely observed in epithelium from palpebral-derived cysts (e). turn interaction, which could be sufficient to maintain a well-defined cell boundary. That this may actually occur in maintaining the limbal-conjunctival epithelial boundary is suggested by recent data from Ljubimov et al, 25 who showed that the basement membranes of the corneal and limbal epithelia contain a specific alpha 5 isoform of type IV collagen, which is absent in the conjunctival basement membrane. Whether this or other differences of the anterior ocular basement membrane are indeed responsible for

10 532 Investigative Ophthalmology & Visual Science, March 1996, Vol. 37, No. 4 maintaining the limbal-conjunctival boundary is a subject that clearly deserves further investigation. Goblet Cell Formation Conjunctival goblet cell production has been tied closely to the degree of vascularization of the underlying stroma. The normal conjunctival stroma underlying the goblet cell-rich epithelium is highly vascularized, whereas the corneal stroma underlying an epithelium devoid of goblet cells is avascular. That vascularization may influence goblet cell formation is suggested by the finding that when the vasculature is replaced in human conjunctival flaps by an avasculardense fibrous scar, goblet cell density is markedly reduced or lost. 2 Furthermore, when conjunctival epithelium migrates over corneal stroma after a total corneal epithelial loss, the elaboration of goblet cells correlates with the presence of vessels. One interpretation of these data is that the absence of goblet cells in corneal epithelium is secondary to a lack of a stromal blood supply. However, our studies argue strongly against this. We found that corneal epithelial cells implanted in a vascular-rich stroma elaborate an epithelium that is devoid of goblet cells, thus indicating that vascularization alone is not sufficient for goblet cell formation. Our data are consistent with previous studies concerning the response of the human corneal epithelium to inflammation. 26 In chronic keratitis, the corneal stroma becomes highly vascularized, with extension of the limbal capillary plexus subepithelially, resulting in the presence of vascular profiles between the "plywood-like" stroma. However, the corneal epithelium overlying this inflammatory-induced, conjunctival-like stroma maintains its original phenotype and does not elaborate goblet cells. Furthermore, in recent studies of spontaneous corneal neovascularization in nude mice, corneal epithelium maintained on a highly vascularized stroma for more than 6 weeks showed no evidence of any morphologic changes. 27 These observations are entirely consistent with the idea that the corneal epithelium lacks the precursor^) to the goblet cell. In conclusion, we have provided in vivo evidence to support our earlier suggestion 8 that the corneallimbal lineage is distinct from the conjunctival epithelial lineage. We have also shown that goblet cell precursors reside exclusively in the conjunctival epithelium, thus providing new insights into conjunctival development. Finally, our description of the athymic mouse as a model for conjunctival development will be particularly useful for the clonal analysis of keratinocyte and goblet cell differentiation potential, and for investigating ocular mucin formation and release. KeyWords cell lineages, conjunctival goblet cells, epithelial differentiation, epithelial stem cells, transdifferentiation References 1. Friedenwald JS. Growth pressure and metaplasia of conjunctival and corneal epithelium. Doc Ophthalmol. 1951;5/6: Tseng SCG, Hirst LW, Farazdaghi M, Green WR. Goblet cell density and vascularization during conjunctival transdifferentiation. Invest Ophthalmol Vis Sri. 1984; 25: Maumenee AE, Scholz RO III. The histopathology of the ocular lesions produced by the sulfur and nitrogen mustards. Bull Johns Hopkins Hosp. 1948; 82: Thoft RA, Friend J, Murphy HS. Ocular surface epithelium and corneal vascularization in rabbits. I. The role of wounding. Invest Ophthalmol Vis Sri. 1979; 18: Liu SH, Tagawa U, Pendergast RA, Franklin RM, Silverstein AM. Secretory component of IgA, a marker for differentiation of ocular epithelium. Invest Ophthalmol Vis Sri. 1981;20: Doran TI, Vidrich A, Sun T-T. Intrinsic and extrinsic regulation of the differentiation of skin, corneal and esophageal epithelial cells. Cell. 1980; 22: Lavker RM, Sun T-T. Rapid modulation of epidermal differentiation by the external environment. / Invest Dermatol 1983; 80: Wei Z-G, Wu RL, Lavker RM, Sun T-T. In vitro growth and differentiation of rabbit bulbar, fornix and palpebral conjunctival epithelia: Implications on conjunctival epithelial transdifferentiation and stem cells. Invest Ophthalmol Vis Sri. 1993;34: Wei Z-G, Cotsarelis G, Sun T-T, Lavker RM. Labelretaining cells are preferentially located in fornical epithelium: Implications for conjunctival epithelial homeostasis. Invest Ophthalmol Vis Sri. 1994; 36: Johnson WC. Histochemistry and special stains. In: Pathology of the Skin. Farmer ER, Hood AF. Norwalk, CT: Appleton & Lange; 1990: Ghadially FN. Ultrastructural Pathology of the Cell and Matrix. London: Butterworths; 1982: Gipson IK, Yankauckas M, Spurr-Michaud SJ, Tisdale AS, Rinehart W. Characteristics of a glycoprotein in the ocular surface glycocalyx. Invest Ophthalmol Vis Sri. 1992;33: Watanabe H, Fabricant M, Tisdale AS, Spurr-Michaud SJ, Lindberg K, Gipson IK. Human corneal and conjunctival epithelial produce a mucin-like glycoprotein for the apical surface. Invest Ophthalmol Vis Sri. 1995; 36: Thoft RA, Friend J. Biochemical aspects of contact lens wear. Am J Ophthalmology. 1975; 80: Thoft RA, Friend J. Biochemical transformation of regenerating ocular surface epithelium. Invest Ophthalmol Vis Sri. 1977; 16: Shapiro MS, Friend J, Thoft RA. Corneal re-epithelialization from the conjunctiva. Invest Ophthalmol Vis Sri. 1981;21: Kinoshita S, Friend J, Thoft RA. Biphasic cell proliferation in transdifferentiation of conjunctival to corneal

11 Modulation of Corneal, limbal, and Conjunctiva! Keratinocytes 533 epithelium in rabbits. Invest Ophthalmol Vis Sci. 1983;24: Kinoshita S, Friend J, Kiorpes TC, Thoft RA. Keratinlike proteins in corneal and conjunctival epithelium are different. Invest Ophthalmol Vis Sci. 1983a; 24: Buck RC. Ultrastructure of conjunctival epithelium replacing corneal epithelium. Curr Eye Res. 1986; 5: Huang AJW, Tseng SCG, Kenyan KR. Morphogenesis of rat conjunctival goblet cells. Invest Ophthalmol Vis Sci. 1988;29: Tseng SCG, Hirst LW, Farazdaghi M, Green WR. Inhibition of conjunctival transdifferentiation by topical retinoids. Invest Ophthalmol Vis Sci. 1987;28: Tseng SCG, Farazdaghi M, Rider AA. Conjunctival transdifferentiation induced by systemic vitamin A deficiency in vascularized rabbit corneas. Invest Ophthalmol Vis Sci. 1987a; 28: Rousselle P, Aumailley M. Kalinin is more efficient than laminin in promoting adhesion of primary keratinocytes and some other epithelial cells and has a different requirement for integrin receptors. / Cell Biol. 1994;125: Khosla J, Correa MT, Sannes PL. Heterogeneity of sulfated microdomains within basement membranes of pulmonary airway epithelium. Am J Respiratory Cell MolBiol. 1994;10: Ljubimov AV, Burgexon RE, Betkowski RJ, Micheal AF, Sun T-T, Kenney MC. Human corneal basement membrane heterogeneity: Topographical differences in the expression of type FV collagen and laminin isoforms. Lab Invest. 1995; 72: Spencer WH. Ophthalmic Pathology: An Atlas and Textbook. Philadelphia: WB Saunders; 1985: Kaminska GM, Niederkown JY. Spontaneous corneal neovascularization in nude mice. Local imbalance between angiogenic and anti-angiogenic factors. Invest Ophthalmol Vis Sci. WB Saunders; 1993;34: