Isoforms of DeltaNp63 and the migration of ocular limbal cells in human corneal regeneration

Abstract

The p63 gene generates transactivating and N-terminally truncated transcripts (DeltaNp63) initiated by different promoters. Alternative splicing gives rise to three different C termini, designated alpha, beta, and gamma. In the ocular epithelium, the corneal stem cells, which are segregated in the basal layer of the limbus, contain the alpha isoform but not beta or gamma. Holoclones derived from the limbus are rich in alpha, meroclones contain little, and paraclones contain none. In normal resting corneal epithelium, p63 of all isoforms is absent. Upon corneal wounding, cells originating from the limbus and containing alpha migrate progressively through the epithelium of the peripheral and central cornea. In the absence of an attached limbus, no alpha isoform appears in the corneal epithelium. When migrating cells containing the alpha isoform appear in the wounded corneal epithelium, they are confined to the basal layer, but the suprabasal cells, not only of the cornea but of the limbus as well, contain mRNA encoding beta and gamma. These data support the concept that the alpha isoform of p63 is necessary for the maintenance of the proliferative potential of limbal stem cells and their ability to migrate over the cornea. The beta and gamma isoforms, being suprabasal and virtually absent from the resting limbus, are not stem cell markers but are likely to play a role in epithelial differentiation specifically during the process of corneal regeneration.

Fig. 1.

Expression of p63 in resting and activated corneas. (A) 4A4 mAb immunofluorescent staining of resting (a and b), activated (c and d), limbal (a and c), and corneal (b and d) epithelium. (B) Progressive appearance of p63 in corneal epithelium. Resting corneas were placed in tissue culture after retrieval from donors. At different incubation time (hours indicated in each frame), specimens were immunostained with 4A4 mAb. Arrows indicate the limbus–cornea border. At time 0, clusters of p63⁺ cells were observed only in the limbal basal layer (asterisks). At 12 h, the entire limbal basal layer expressed p63. Note the progressive appearance of p63⁺ cells from the peripheral cornea to the central cornea (24–72 h). (C) p63 appears in the corneal epithelium as a result of cell migration from the limbus. The central cornea and the limbal ring of resting corneas were surgically separated with a 5-mm corneal trephine immediately after retrieval (a) and placed in tissue culture for 5 days. Samples were then immunostained with 4A4 mAb. Note that p63⁺ cells were present in the limbal ring (b) but not in the separated central cornea (c). When the two were not separated, the cornea (e), like its associated limbus (d), expressed p63.

Fig. 2.

RT-PCR analysis in cells cultured from resting and activated limbus and cornea. Semiquantitative RT-PCR analysis was performed by using primers specific for each of the p63 isoforms (Table 1). All three isoforms of ΔNp63 are present in cells cultured from the resting limbus but appear in the cornea only after activation. E and F show quantitation of PCR products shown in A, B, and D, respectively. β-Actin (βact) has been used as a control. Note the absence of all TAp63 isoforms.

Fig. 3.

Cytological detection of ΔNp63α,-β, and -γ transcripts in resting vs. activated limbus and cornea. Sections were processed for in situ hybridization with antisense digoxigenin-labeled cRNAs specific for each of the ΔNp63 isoforms. Note the abundant expression of β and γ transcripts in activated but not in resting epithelia. The dotted line underlines the basal layer of activated limbal and corneal epithelia.

Fig. 4.

Specificity of the antiserum raised against p63α. Parallel cultures of 3T3 cells were transfected with cDNA encoding either ΔNp63α (lanes 1), ΔNp63β (lanes 2), or ΔNp63γ (lanes 3). Cell extracts prepared from transduced 3T3 cells were fractionated on 7.5% SDS-polyacrylamide gels, transferred to poly(vinylidene difluoride) filters, and immunostained. 4A4 mAb (A) detects all three isoforms, whereas or p63α-specific IgG detects only the α isoform (B).

Fig. 5.

Immunodetection of ΔNp63α in resting and activated limbus and cornea. (A) Double immunofluorescence of sections of resting and activated limbus stained with 4A4 mAb (green) and p63α-specific IgG (red). The yellow color in the “merge” frames indicates cells stained with both antibodies. Cells stained green alone express β and γ ΔNp63 but not the α isoform. The two series of frames indicated as resting limbus are from different experiments representative of the lowest (Left) and highest (Right) number of α-expressing cells, respectively. (B) Z-stack analysis of 6-μm sections of activated limbus and cornea immunostained with 4A4 mAb and α-specific IgG. Images were obtained with a LSM510 confocal microscope (Zeiss) and Z-stack analyzed with the LSM510 Analyzer. Most suprabasal cells express some isoforms of ΔNp63 but not α.

Fig. 6.

Identification of holoclones by their content of ΔNp63α. One hundred cells obtained from primary limbal cultures were plated in 100-cm dishes and cultured for 1 week. Colonies were then examined under an Axiovert 200 M microscope (Zeiss); large round colonies with smooth and regular borders and formed entirely by small cells with scarce cytoplasm were classified as holoclones. Large colonies formed by small cells but showing irregular borders and/or with areas containing large cells were classified as meroclones. Small colonies with wrinkled and irregular borders and formed by large cells were classified as paraclones. Double immunofluorescence was performed on paraformaldehyde-fixed colonies with 4A4 mAb (green) and p63α-specific IgG (red). The yellow color in the “merge” frames indicates cells stained with both antibodies. The three clonal types are most clearly distinguished by the presence or the absence of the α isoform.

Fig. 7.

Clonogenic and proliferative capacities and p63 content of cells cultured from resting and activated limbus and cornea. (A–D) Clonogenic ability. One thousand keratinocytes obtained from primary cultures established from resting and activated limbus and cornea were cultivated for 12 days and stained with Rhodamine B. Although resting corneal cells generated only few aborted colonies (B), resting limbal cells (A) and activated limbal (C) and corneal (D) cells had a comparable clonogenic ability. (E) Proliferative capacity. Keratinocytes were then serially cultivated, and the number of cell generations was calculated. Although resting corneal cells could not be passaged (blue), resting limbal (yellow), activated limbal (red), and activated corneal cells (green) underwent a comparable number of cell doublings. (F) p63 content by Western analysis. Cell extracts were prepared from limbal (lanes 1 and 3) and corneal (lanes 2 and 4) cultures established from resting (lanes 1 and 2) and activated (lanes 3 and 4) corneas. Equal amounts of protein were fractionated on 7.5% SDS-polyacrylamide gels, transferred to poly(vinylidene difluoride) filters, and immunostained with 4A4 mAb. ΔNp63α, β, and γ were detected in limbal but not in corneal cells cultivated from resting corneas and in both limbal and corneal cells cultured from activated corneas.