Isoforms of ΔNp63 and the migration of ocular limbal cells in human corneal regeneration

Abstract

The p63 gene generates transactivating and N-terminally truncated transcripts (⌬Np63) initiated by different promoters. Alternative splicing gives rise to three different C termini, designated ␣, ␤, and ␥. In the ocular epithelium, the corneal stem cells, which are segregated in the basal layer of the limbus, contain the ␣ isoform but not ␤ or ␥. Holoclones derived from the limbus are rich in ␣, 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 ␣ migrate progressively through the epithelium of the peripheral and central cornea. In the absence of an attached limbus, no ␣ isoform appears in the corneal epithelium. When migrating cells containing the ␣ 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 ␤ and ␥. These data support the concept that the ␣ 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 ␤ and ␥ 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. The corneal epithelium provides an ideal experimental system in which to distinguish keratinocyte stem and TA cells (8). Corneal stem cells are segregated in the basal layer of the limbus, which is the zone encircling the cornea and separating it from the bulbar conjunctiva. TA cells that migrate from the limbus form the corneal epithelium (8). That the limbus is the site of stem cell precursors of the corneal epithelium is clear for several reasons: (i) the basal layer of the limbus lacks keratin 3 (a marker for corneal differentiation), whereas limbal suprabasal layers and all layers of the corneal epithelium express keratin 3 (9); (ii) the limbus contains slow-cycling cells and holoclone-forming cells, but the corneal epithelium does not (7, 10); (iii) the corneal epithelial cells are not self-sustaining; they divide only a few times during their migration from the limbus to the central cornea (11); (iv) restoration of destroyed limbal͞corneal epithelium requires limbal transplantation (12) or grafts of autologous limbal cultures (13-15).The gene with the most striking effects on the development of stratified epithelia is p63 (16-19). Ablation of the p63 gene in mice results in the absence of these epithelia (17,18). In humans, mutations of the p63 gene cause disorders of the epithelia and of nonepithelial structures whose development depends on the epithelial functions (20). The p63 gene generates six isoforms (21). The transactivating isoforms are generated by the activity of an upstream promoter; the ⌬N isoforms are produced from a downstream intronic promoter and lack the transactivation domain. For both transcripts,...

Stem cells of the adult cornea: From cytometric markers to therapeutic applications

“…It was also shown that antibodies detecting all isoforms of p63 identified differentiated cells as well (66). The isoform DNP63a was later shown to identify epithelial stem cells, whereas b and c isoforms have been shown to promote epithelial cell differentiation (67,68). At present, DNp63a is considered as a reliable marker of both resting and activated limbal epithelial stem cells (47).…”

Niche regulation of corneal epithelial stem cells at the limbus

“…In contrast, the SC-containing limbal epithelium has a high proliferative potential in different cultures [12][13][14][15], and their in vitro proliferation is resistant to the inhibition by tumor-promoting phorbol esters [13,16,17]. Furthermore, limbal basal epithelial cells express cytokeratin 19 [18], and integrin a9 [10,19], and preferentially express such progenitor markers as p63 [20], especially its ∆Np63a isoform [21,22], Bcrp1/ABCG2 [10,[23][24][25], and N-cadherin [26].…”

CD38 and CD157 Ectoenzymes Mark Cell Subsets in the Human Corneal Limbus

“…Corneal CD157 appears as a doublet with a M r of 42-to 45-kDa, suggesting the existence of different isoforms, likely produced by alternative glycosylation (35) Epithelium regenerates during wound repair by migration of cells from the corneal limbus (1,28). However, an open issue in corneal regeneration is the lack of a dependable surface marker of the limbal stem cell population (41).…”

“…The protein was more apparent in the cells at the center of each cluster, which in turn tended to accumulate at the palisades of Vogt (see Figure 4A), a region of the limbus that harbors the highest concentration of stem cells (1,27). Nuclear p63 antigen is a putative marker of limbal stem cells (28) and, according to merged-double staining, p63 + was coexpressed with CD157 within the basal limbal layer (see Figure 4C). CK19 is expressed by limbal and peripheral corneal epithelial cells (29), suggesting that CD157 and CK19 may express partially overlapped patterns.…”