The Labelling Index Is Not Always Reliable. Discrepancies Between the Labelling Index and the Mitotic Rate In the Rat Corneal Epithelium After Intraperitoneal and Topical Administration of Tritiated Thymidine and Colcemid

Abstract. Many cell kinetic studies are based on the assumption that tritiated thymidine injected into an animal is available for incorporation into DNA for only a short time, and that it labels all cells in the S phase. the present study indicates that this is not the fact for the rat corneal epithelium. the labelling index (LI) declines considerably from the limbal area to the central cornea, while the mitotic rate is almost constant all over the corneal epithelium. the LI should therefore not be used as the only criterion in the assessment of proliferation rate.     

Circadian variations in the DNA synthesis of the rat corneal epithelium. A study using double labelling with tritiated thymidine

A prominent circadian rhythm was found in the labelling indices (LI) of the peripheral rat corneal epithelium and of the adjacent conjunctival epithelium, while almost no diurnal variation was found in the central area. Application of a double labelling technique indicated that there are rhythmic pulses of high and low influx of cells into the S phase and similar pulses of efflux of cells from the S phase. Results of the study indicate that there are different cohorts of cycling cells all over the rat corneal epithelium. Cells belonging to a rapidly proliferating cohort are observed in the peripheral cornea. There is a gradual reduction in the fraction of labelled DNA-synthesizing cells towards the centre. The considerably lower fraction of cells taking up tritiated thymidine (3H)TdR in the central cornea may be due to a higher fraction of basal cells having reached higher levels of differentiation. This may result in a shift from the salvage to the de novo pathway. The slowly proliferating cohort seems to have a prolonged S phase duration and displays practically no diurnal variation in the LI. The DNA-synthesizing cells belonging to this latter cohort probably use the salvage pathway for DNA synthesis resulting in uptake of (3H)TdR all over the cornea. The LI is thus not a reliable indicator of cell proliferation in the corneal epithelium, due both to the heterogeneity of the cell proliferation, and in particular due to the lack of labelling of the centrally located DNA-synthesizing cells. To what extent these properties may also be present in other proliferating tissues with different levels of differentiations, may be questioned.     

Optimal conditions for immunohistochemical determination of the in vitro DNA synthesis labelling index with bromodeoxyuridine in head and neck cancer

The in vitro DNA synthesis labelling index was assessed immunohistochemically in 24 freshly obtained specimens of head and neck cancer using bromodeoxyuridine (BrdUrd) as the DNA precursor to determine the influence of BrdUrd concentration on labelling index (LI). Initially, tumour fragments were incubated in varying concentrations of BrdUrd from 2 to 100 microM for 2 h, and BrdUrd was detected with an anti-BrdUrd monoclonal antibody using immunoperoxidase labelling. There was a dose-response gradient with mean LI varying from 1.6% at 2 microM BrdUrd to 8.8% at 100 microM. The concentration-response gradient best fit a quadratic model when LI was plotted against log BrdUrd concentration (r = 0.65, P less than 0.0001). Eleven additional tumours were then studied to determine whether LI increased for BrdUrd concentrations above 100 microM. The mean LI at 125 microM and at 150 microM in these 11 tumours did not differ from the value at 100 microM, suggesting a plateau at this level. The gradient effect accounted for 17% of the variance in LI, while 60% of the variance was explained by between tumour differences. Within individual tumours, three response patterns were observed: (i) LI rose at a constant rate to the highest concentration tested (n = 8), (ii) the LI plateaued or declined at high BrdUrd concentrations (n = 6); and (iii) there was a biphasic slope slope in which the rate of rise in the LI increased at the higher BrdUrd concentrations (n = 2). The data show that BrdUrd concentration is an important variable in the immunohistochemical assessment of the in vitro LI in head and neck cancer.     

Ki-67 labelling index in human brain tumours

The proliferative potential in 157 brain tumours was investigated using Ki-67 labelling index (Ki-67LI). There were 46 patients with low grade gliomas (Al & All), 82 with high grade gliomas (AIII & AIV) and 29 with metastatic tumours. Tumour fragments used for assessment of Ki-67LI were fixed in formalin. Ki-67 antigen was visualised on paraffin sections using DAKO Rabbit Anti-Human Ki-67 antigen. The Ki-67LI was calculated as the percentage of Ki-67 labelled cells. The tumours showed variability in the Ki-67LI values. Significantly higher mean Ki-67LI was found for highly malignant (AIII & AIV) than for low grade gliomas (Al & All). For metastatic tumours, the mean values of Ki-67LI were significantly higher than for gliomas. Moreover, Ki-67LI of metastatic tumours were significantly higher than for high grade gliomas.     

Labelling index of human squamous cell carcinomas. Comparison of in vivo and in vitro labelling methods.

In vivo and in vitro labelling has been compared in sixteen human solid squamous cell carcinomas (ENT). The median in vivo/in vitro LI ratio was 1-2, but for two-thirds of the patients it was only 1-1, suggesting a slight LI underestimation in vitro. Two factors can possibly explain the divergences: heterogeneity from one biopsy to another in the same tumour, and lower mean grain count in the deep cell layers of the in vitro labelled tumour samples. Therefore, the fact we did not find a good agreement between in vivo and in vitro data for one-third of the tumours points out that one must be cautious in considering in vitro LI as a valid result for a given patient. However, even if the in vitro LI leads to a certain underestimate, it can provide useful data.     

Reactive oxygen species (ROS)-generating oxidases in the normal rabbit cornea and their involvement in the corneal damage evoked by UVB rays

The corneas of albino rabbits were irradiated (5 min exposure once a day) with UVB rays (312 nm) for 4 days (shorter procedure) or 8 days (longer procedure). The eyes were examined microbiologically and only the corneas of sterile eyes or eyes with non-pathogenic microbes were employed. Histochemically, the activities of reactive oxygen species (ROS)-generating oxidases (xanthine oxidase, D-amino acid oxidase and alpha-hydroxy acid oxidase) were examined in cryostat sections of the whole corneas. Biochemically, the activity of xanthine oxidoreductase/xanthine oxidase was investigated in the scraped corneal epithelium. UVB rays significantly changed enzyme activities in the corneas. In comparison to the normal cornea, where of ROS-generating oxidases only xanthine oxidase showed significant activity in the corneal epithelium and endothelium, D-amino acid oxidase was very low and alpha-hydroxy acid oxidase could not be detected at all, in the cornea repeatedly irradiated with UVB rays, increased activities of xanthine oxidase and D-amino acid oxidase were observed in all corneal layers. Only after the longer procedure the xanthine oxidase and D-amino acid oxidase activities were decreased in the thinned epithelium in parallel with its morphological disturbances. Further results show that the xanthine oxidase/xanthine oxidoreductase ratio increased in the epithelium together with the repeated irradiation with UVB rays. This might suggest that xanthine dehydrogenase is converted to xanthine oxidase. However, in comparison to the normal corneal epithelium, the total amount of xanthine oxidoredutase was decreased in the irradiated epithelium. It is presumed that xanthine oxidoreductase might be released extracellularly (into tears) or the enzyme molecules were denatured due to UVB rays (particulary after the longer procedure). Comparative histochemical and biochemical findings suggest that reactive oxygen species-generating oxidases (xanthine oxidase, D-amino acid oxidase) contribute to the corneal damage evoked by UVB rays.     

Biosynthesis of glycosaminoglycans by the separated tissues of the embryonic chick cornea

Corneal tissues (epithelium, endothelium, and stroma) were isolated from chick embryos at 14, 17, and 20 days of incubation and immediately labeled in vitro with d-[6-³H]glucosamine and H₂³⁵SO₄. Amount of label incorporated into each type of glycosaminoglycan or into glycopeptides was determined by specific degradative techniques, in conjunction with gel filtration chromatography. Results suggested that corneal epithelium synthesized little, if any, corneal keratan sulfates, but that corneal endothelium may have synthesized small amounts of corneal keratan sulfates. Nearly all corneal keratan sulfates were derived from the stroma. Corneal heparan sulfates appeared to be derived predominantly from corneal epithelium at later stages of development. Corneal endothelium contributed large proportions of the hyaluronic acids of the cornea. Only epithelium produced a large proportion of sulfated glycoproteins. In addition, epithelium synthesized a large proportion of a sulfated, high molecular weight polysaccharide which was resistant to treatments degrading known types of glycosaminoglycans. Each corneal tissue may not only affect corneal morphogenesis directly by contributing a unique spectrum of glycosylated proteins to the extracellular matrix, but also may regulate the extracellular matrix composition indirectly by modulating the biosynthetic activities of the other corneal tissues.     

Double labelling of cells with tritiated thymidine and bromodeoxyuridine reveals a circadian rhythm-dependent variation in duration of DNA synthesis and S phase flux rates in rodent oral epithelium

Abstract. We describe a double labelling method for estimating the duration of DNA synthesis (Ts) and the flux of cells into and from the S phase of the cell cycle, based on labelling with tritiated thymidine ([³H]TdR) followed by bromodeoxyuridine (BrdU) and combining immunohistological detection of BrdU with conventional autoradiography. In practice, the change in size of a window of double labelled cells occurs as the time interval between the two labels increases. In mouse tongue epithelium there is a marked circadian variation in the number of cells in DNA synthesis. From 0900 to 1500 h this labelling index (LI) falls, but from 2100 to 0300 h it increases. Our results show that the circadian decrease in LI is associated with a short Ts (5·8 ± 0·3 h), a high S phase efflux and an initially low influx of cells from G_: into S. Conversely, the rising circadian LI is associated with a longer Ts (9.4 ± 0.1 h), an initially low efflux and a moderate to high influx. Two time-points exist on the circadian LI curve when influx and efflux rates change abruptly. At 0100 h the efflux rate rises from low (5 cells %/h) to high (15–16 cells %/h) and simultaneously the influx rate changes from high to low. Similarly at 1300–1400 h, efflux rate falls from high (19–20 cells %/h) to low (4–8 cells %/h) values and influx rates change from low to high. This double labelling method has revealed that the duration of DNA synthesis varies across the circadian cycle, as do influx and efflux values which generally fall within a discrete range of high or low values. The timing of the changes in flux suggests the presence of two 'control' points on the circadian LI cycle that were previously unrecognized.     

Double labelling of cells with tritiated thymidine and bromodeoxyuridine reveals a circadian rhythm-dependent variation in duration of DNA synthesis and S phase flux rates in rodent oral epithelium

We describe a double labelling method for estimating the duration of DNA synthesis (Ts) and the flux of cells into and from the S phase of the cell cycle, based on labelling with tritiated thymidine [( 3H]TdR) followed by bromodeoxyuridine (BrdU) and combining immunohistological detection of BrdU with conventional autoradiography. In practice, the change in size of a window of double labelled cells occurs as the time interval between the two labels increases. In mouse tongue epithelium there is a marked circadian variation in the number of cells in DNA synthesis. From 0900 to 1500 h this labelling index (LI) falls, but from 2100 to 0300 h it increases. Our results show that the circadian decrease in LI is associated with a short Ts (5.8 +/- 0.3 h), a high S phase efflux and an initially low influx of cells from G1 into S. Conversely, the rising circadian LI is associated with a longer Ts (9.4 +/- 0.1 h), an initially low efflux and a moderate to high influx. Two time-points exist on the circadian LI curve when influx and efflux rates change abruptly. At 0100 h the efflux rate rises from low (5 cells %/h) to high (15-16 cells %/h) and simultaneously the influx rate changes from high to low. Similarly at 1300-1400 h, efflux rate falls from high (19-20 cells %/h) to low (4-8 cells %/h) values and influx rates change from low to high. This double labelling method has revealed that the duration of DNA synthesis varies across the circadian cycle, as do influx and efflux values which generally fall within a discrete range of high or low values. The timing of the changes in flux suggests the presence of two 'control' points on the circadian LI cycle that were previously unrecognized.     

Localization of DNA-synthesizing cells and cell proliferation pattern in developing proventricular (glandular stomach) epithelium of embryonic and hatched chickens

Localization of DNA-synthesizing cells in the developing proventricular (glandular stomach) epithelium of embryonic and hatched chickens was investigated. DNA-synthesizing cells were scattered throughout the proventricular epithelium during all developmental stages studied. The results indicate that there is no clear proliferative zone in the proventricular epithelium of the chicken. The labeling indices (LI) of proventricular epithelial cells were measured. On the 6.5th day of incubation, the LI of glandular epithelium reached 29.5 ± 1.5%. the highest value of all the stages studied. This extremely rapid cell proliferation can be considered to be a driving force for the elongation of the proventricular glands during the following stages. Just after hatching, the LI of both the glandular and luminal (non-glandular) epithelia significantly increased from those on the 18th day of incubation. It is suggested that the rise in LI possibly reflected proventricular growth to fit in the change in the method of nourishment after hatching. In 2 week old chickens, the LI of both the glandular and luminal epithelia were reduced to approximately 1%. The active production of embryonic chicken pepsinogen in all glandular epithelial cells of the embryonic chicken revealed that proliferation and differentiation are not necessarily exclusive during the embryonic stages of proventricular development.     

The lens organizes the anterior segment: specification of neural crest cell differentiation in the avian eye

During the development of the anterior segment of the eye, neural crest mesenchyme cells migrate between the lens and the corneal epithelium. These cells contribute to the structures lining the anterior chamber: the corneal endothelium and stroma, iris stroma, and trabecular meshwork. In the present study, removal of the lens or replacement of the lens with a cellulose bead led to the formation a disorganized aggregate of mesenchymal cells beneath the corneal epithelium. No recognizable corneal endothelium, corneal stroma, iris stroma, or anterior chamber was found in these eyes. When the lens was replaced immediately after removal, a disorganized mass of mesenchymal cells again formed beneath the corneal epithelium. However, 2 days after surgery, the corneal endothelium and the anterior chamber formed adjacent to the lens. When the lens was removed and replaced such that only a portion of its anterior epithelial cells faced the cornea, mesenchyme cells adjacent to the lens epithelium differentiated into corneal endothelium. Mesenchyme cells adjacent to lens fibers did not form an endothelial layer. The cell adhesion molecule, N-cadherin, is expressed by corneal endothelial cells. When the lens was removed the mesenchyme cells that accumulated beneath the corneal epithelium did not express N-cadherin. Replacement of the lens immediately after removal led to the formation of an endothelial layer that expressed N-cadherin. Implantation of lens epithelia from older embryos showed that the lens epithelium maintained the ability to support the expression of N-cadherin and the formation of the corneal endothelium until E15. This ability was lost by E18. These studies provide evidence that N-cadherin expression and the formation of the corneal endothelium are regulated by signals from the lens. N-cadherin may be important for the mesenchymal-to-epithelial transformation that accompanies the formation of the corneal endothelium.     

In vivo labelling with halogenated pyrimidines of squamous cell carcinomas and adjacent non-involved mucosa of head and neck region

The frequency and distribution of labelled cells were studied immunohistochemically in 37 squamous cell carcinomas (SCC) of head and neck after in vivo infusion of IdUrd and BrdUrd. Tumours were classified according to their labelling patterns. Low and moderate grade SCC consisted of tumour islands separated by interstitial tissue. In some tumours labelled cells only appeared near the basal layer while in others proliferative cells were evenly distributed within the neoplastic island. In anaplastic carcinomas labelled cells were distributed either randomly or around blood vessels (cord structures). While the basal layer in adjacent normal epithelium contained very few labelled cells (LI = 1.6 ± 0.2%), the LI of basal cells in tumour islands were much higher than the average LI of the tumour (47.2 ± 2.8% and 23.8 ± 1.6%, respectively). In patients who had received cytotoxic therapy up to two months before the biopsy, the LI in the basal layer of normal epithelium was 19.0 ± 3.5%. In sequential biopsies obtained 1–2 weeks after the infusion of IdUrd and BrdUrd some labelled tumour cells were found in necrotic foci and in pearl structures. Additionally, in six tumours, we found areas of cells labelled with IdUrd alone, even though the IdUrd infusion had been followed by a BrdUrd infusion 1 h later. This is in agreement with the phenomenon of intermittent tumour blood flow described earlier in experimental tumours.     

Changes in the DNA labelling index after beta irradiation of the mouse epidermis

This study looked at the changes in the interfollicular DNA labelling index (LI) with time after strontium-90/yttrium-90 beta irradiation of approximately 100 mm2 of mouse flank skin, after a dose of 100 Gy which produces transitory moist desquamation. Within 24 hr of such a dose the LI of the irradiated area was essentially zero (0.07 +/- 0.03%), whilst those of the side area and of the control area were 15.0 +/- 2.6% and 21.4 +/- 2.7%, respectively. The LI of the side and the control areas then fell within 3-5 days to approximately 4% and approximately 2% respectively, whilst that of the irradiated area rose rapidly to a peak value of 30.2 +/- 1.7% at 10 days post-irradiation. There was a 20% reduction in the diameter of the area with detectable radiation damage within 5 days, and this is primarily due to cell proliferation and migration from the unirradiated margins of the field. In contrast, between days 10 and 20 the major source of repopulation is probably derived from local migration and proliferation of surviving hair follicle basal cells within the irradiated field.     

A comparison of lyophilized amniotic membrane with cryopreserved amniotic membrane for the reconstruction of rabbit corneal epithelium

Many researchers have employed cryopreserved amniotic membrane (CAM) in the treatment of a severely damaged cornea, using corneal epithelial cells cultured on an amniotic membrane (AM). In this study, two Teflon rings were made for culturing the cells on the LAM and CAM, and were then used to support the AM, which is referred to in this paper as an Ahn’s AM supporter. The primary corneal epithelial cells were obtained from the limbus, using an explantation method. The corneal epithelium could be reconstructed by culturing the third-passage corneal epithelial cells on the AM. A lyophilized amniotic membrane (LAM) has a higher rate of graft take, a longer shelf life, is easier to store, and safer, due to gamma irradiation, than a CAM. The corneal epithelium reconstructed on the LAM and CAM, supported by the two-Teflon rings, was similar to normal corneal epithelium. However, the advantages of the LAM over that of the CAM make the former more useful. The reconstruction model of the corneal epithelium, using AM, is considered as a goodin vitro model for transplantation of cornel epithelium into patients with a severely damaged cornea.     

Basonuclin in murine corneal and lens epithelia correlates with cellular maturation and proliferative ability

Basonuclin is a zinc finger protein with highly restricted tissue distribution. It has been found in abundance only in keratinocytes of stratified epithelia and the germ cells of the testis and ovary. We studied the expression pattern of basonuclin in relation to cellular proliferation and differentiation in murine corneal and lens epithelia, two self-renewing tissues in the eye which contain cells that proliferate throughout life. Mouse corneal and lens epithelial cells at various stages of development were labeled with BrdU for 90 min to detect cells in S phase and to establish proliferative rates. Whole eyes of mouse or rat were processed for frozen sections and cellular basonuclin was detected by either a rabbit antimouse- or a rabbit anti-human-basonuclin antibody. Basonuclin was expressed in virtually all cells in the basal layer of corneal epithelium and in the pre-equatorial lens epithelium, the respective proliferative compartments of adult corneal and lens epithelia. Basonuclin expression in corneal epithelium began at post-natal life day 4, first in a few cells and then spread to virtually all basal cells at day 20. Basonuclin was consistently absent in limbal epithelium. Lens basonuclin, which was detected earlier than that of the cornea, was confined to the pre-equatorial epithelium and was absent in equatorial cells that expressed p57KIP2, an early differentiation marker for these cells. An important distinction between corneal and lens basonuclin is that the former is predominantly nuclear whereas the latter cytoplasmic.     

Reconstruction of a rabbit corneal epithelium on a lyophilized amniotic membrane using tilting air-liquid interface culture followed by tilting submerged culture

Herein, we reconstructed a rabbit corneal epithelium on a lyophilized amniotic membrane (LAM) using a modified version of two Teflon rings (the Ahn’s supporter). We compared the corneal epithelial cells we had differentiated in vitro using air-liquid interface (6 days, 12 days) and submerged (6 days, 12 days) cultures and followed a six-day tilting dynamic air-liquid interface culture with a six-day tilting submerged culture. We characterized the reconstructed corneal epithelium using digital photography, histological imaging, and transmission electron microscopy. The reconstructed corneal epithelium created under air-liquid interface culture exhibited a healthier basal corneal epithelial layer than that created under submerged culture. The reconstructed corneal epithelium on the LAM that was produced using the tilting dymanic culture exhibited a healthy basal layer. We therefore proposed that tilting submerged culture not only supplied nutrients from the medium to the corneal epithelial cells on the LAM, but it also removed the horny layer in the upper part of the reconstructed corneal epithelium, presumably by mimicking the effects of blinking. This study demonstrated that corneal epithelium reconstruction on a LAM using a tilting submerged culture after a tilting air-liquid interface culture may be useful not only for allogeneic or autologous transplantation, but also for in vitro toxicological test kits.     

The cell kinetics of the epidermis and follicular epithelium of the rat: variations with age and body site

The skin from rats of differing age was used to quantify variations in the cell kinetics of the epidermis and the follicular epithelium of different body sites. Four parameters were assessed, namely the basal cell density (BCD), the labelling index (LI), the duration of DNA synthesis (ts) and the basal cell turnover time (tT). The BCDs of the epidermis of the dorsum and the upper surface of the foot were similar in rats of 7, 14 and 52 weeks of age, but there was an indication of a progressive decline with increasing age in the BCD of the epidermis of the ear and tail. There were no age-related changes in the length of ts in any of the four body regions. The rate of cell proliferation, as indicated by the values of the LI and tT, was relatively rapid in the epidermis of the dorsum, foot and tail of rats aged 7 weeks (LI greater than 12%; tT less than 80 h). In rats aged 14 weeks this rate of proliferation was maintained in the epidermis of the dorsum. However, in the foot and tail the rate of cell proliferation was decreased (LI less than 10%; tT greater than 85 h). A fall in the rate of proliferation of the epidermis of the dorsum was only seen in 52-week-old animals. In these animals the rates of proliferation in the foot and tail were similar to those at the age of 14 weeks. In the epidermis of the ear there was no appreciable change in the rate of cell proliferation with age. The values of the cell kinetic parameters varied in the different body sites. For example, in 52-week-old animals values for tT were relatively short in the epidermis of the tail and foot and appreciably longer in the epidermis of the dorsum and ear. Considered overall, values for the cell kinetic parameters of the epidermis were comparable with those for the follicular epithelium. The only major differences between the epidermis and the follicular epithelium were in the upper surface of the foot at 7 weeks of age, and in the tail at 7 and 14 weeks of age, where the LI was higher and the tT shorter in the epidermis than in the follicular epithelium. The relevance of the observed age- and body-site-related variations in the cell kinetics of the epidermis are discussed in relation to previously described differential changes in the radiosensitivity of the skin in this strain of rat.     

Proliferative activity of primary breast cancer and of synchronous lymph node metastases evaluated by [3H]-thymidine labelling index

Abstract. The [³H]-thymidine labelling index ([³H]TdR LI) has been used to evaluate and comparatively analyse the proliferative activity of different tumour lesions from the same patient. The analysis was performed on the primary tumour and its synchronous lymph node metastasis from 210 patients operated on for breast cancer. A direct relation was observed between the proliferative activity of the two different lesions (Spearman correlation coefficient = 0–46, P< 00001), but there was considerable scatter amongst the data. The [³H]TdR LI of primary and of metastatic lesions belonged to the same proliferation classes in only 47% of the cases. Higher or lower [³H]TdR LI values, categorized on the basis of the tertiles of the frequency distribution, occurred in the node metastasis than in the primary tumour in an almost similar percentage of the remaining cases. Menopause, receptor status and pathological features did not affect interlesion kinetic patterns. The prognostic role of the proliferative activity of the two different lesions was investigated on 107 patients with stage II tumours homogeneously treated with surgery and systemic adjuvant therapy. Relapse-free survival at 3 years was significantly affected by the proliferative activity of the primary tumour but not by that of the lymph node metastasis.     

Circadian variation in the mitotic rate of the rat corneal epithelium. Cell divisions and migration are analyzed by a mathematical model

A stathmokinetic method was used to study the diurnal variation in the mitotic rate (MR) of the rat corneal epithelium, and in the adjacent conjunctival epithelium. A prominent circadian variation in cell proliferation was observed in both epithelia, both showing almost the same pattern, which may indicate that both tissues are submitted to the same regulatory mechanisms. The average rate of cell renewal during a 24 h period indicated a mean cell renewal time of 12.3 days. This is longer than previously assumed. The MR declined toward the central cornea. Based on the above observations and the known centripetal migration of cells in the corneal epithelium, we have developed a mathematical model showing isomorphism with the renewal of the corneal epithelium.     

Dkk2 plays an essential role in the corneal fate of the ocular surface epithelium

The Dkk family of secreted cysteine-rich proteins regulates Wnt/beta-catenin signaling by interacting with the Wnt co-receptor Lrp5/6. Here, we show that Dkk2-mediated repression of the Wnt/beta-catenin pathway is essential to promote differentiation of the corneal epithelial progenitor cells into a non-keratinizing stratified epithelium. Complete transformation of the corneal epithelium into a stratified epithelium that expresses epidermal-specific differentiation markers and develops appendages such as hair follicles is achieved in the absence of the Dkk2 gene function. We show that Dkk2 is a key regulator of the corneal versus epidermal fate of the ocular surface epithelium.