Expression of pluripotency factors in larval epithelia of the frog Xenopus: Evidence for the presence of cornea epithelial stem cells

Abstract

Understanding the biology of somatic stem cells in self renewing tissues represents an exciting field of study, especially given the potential to harness these cells for tissue regeneration and repair in treating injury and disease. The mammalian cornea contains a population of basal epithelial stem cells involved in cornea homeostasis and repair. Research has been restricted to mammalian systems and little is known about the presence or function of these stem cells in other vertebrates. Therefore, we carried out studies to characterize frog cornea epithelium. Careful examination shows that the Xenopus larval cornea epithelium consists of three distinct layers that include an outer epithelial layer and underlying basal epithelium, in addition to a deeper fibrous layer that contains the main sensory nerve trunks that give rise to numerous branches that extend into these epithelia. These nerves convey sensory and presumably also autonomic innervation to those tissues. The sensory nerves are all derived as branches of the trigeminal nerve/ganglion similar to the situation encountered in mammals, though there appear to be some potentially interesting differences, which are detailed in this paper. We show further that numerous pluripotency genes are expressed by cells in the cornea epithelium, including: sox2, p63, various oct4 homologs, c-myc, klf4 and many others. Antibody localization revealed that p63, a well known mammalian epithelial stem cell marker, was localized strictly to all cells in the basal cornea epithelium. c-myc, was visualized in a smaller subset of basal epithelial cells and adjacent stromal tissue predominately at the periphery of the cornea (limbal zone). Finally, sox2 protein was found to be present throughout all cells of both the outer and basal epithelia, but was much more intensely expressed in a distinct subset of cells that appeared to be either multinucleate or possessed multi-lobed nuclei that are normally located at the periphery of the cornea. Using a thymidine analog (EdU), we were able to label mitotically active cells, which revealed that cell proliferation takes place throughout the cornea epithelium, predominantly in the basal epithelial layer. Species of Xenopus and one other amphibian are unique in their ability to replace a missing lens from cells derived from the basal cornea epithelium. Using EdU we show, as others have previously, that proliferating cells within the cornea epithelium do contribute to the formation of these regenerated lenses. Furthermore, using qPCR we determined that representatives of various pluripotency genes (i.e., sox2, p63 and oct60) are upregulated early during the process of lens regeneration. Antibody labeling showed that the number of sox2 expressing cells increased dramatically within 4 hours following lens removal and these cells were scattered throughout the basal layer of the cornea epithelium. Historically, the process of lens regeneration in Xenopus had been described as one involving transdifferentiation of cornea epithelial cells (i.e., o...

Equine Epidermis: A Source of Epithelial-Like Stem/Progenitor Cells with In Vitro and In Vivo Regenerative Capacities

“…Scale bars represent 50 mm. Color images available online at www.liebertpub.com/scd expression in corneal stem cells [41,42] and circulating keratinocyte progenitor cells [32]. Moreover, the EpSCs in the present study were positive for Casein Kinase 2b, which is reputed to inhibit the development of the mesenchymal morphology and phenotype [37,45], and plays an important role in the functional regulation of epithelial cells [46][47][48].…”

“…S6). Finally, most of the EpSCs as well as several of their more differentiated progeny were positive (nuclear and weaker cytoplasmic signal) for the epithelial stem cell marker p63 [41,42] in adhesion as well as in suspension (Supplementary Fig. S7).…”

Expression of pluripotency factors in echinoderm regeneration

“…The most notable difference between the gut and the RNC is the absence of SoxB1 expression during early stages of intestinal regeneration, whereas it remains expressed at the normal level in the injured radial nerve. This observation suggests that cell dedifferentiation in the gut mesothelium does not require expression of SoxB1 , which is puzzling, as Sox2, the homolog of H. glaberrima SoxB1, is not merely expressed in various vertebrate regeneration models (Christen et al, 2010; Luz-Madrigal et al, 2014; Maki et al, 2009; Perry et al, 2013), but is absolutely required for regeneration (Christen et al, 2010). The functional significance of SoxB1 down-regulation in the regenerating sea cucumber intestine is unknown and can only be determined in future experiments involving forced ectopic over-expression.…”

“…Expression of Yamanaka factors in post-traumatic regeneration has been previously described in various organs of regeneration-competent vertebrates, such fish and amphibians (Christen et al, 2010; Maki et al, 2009; Perry et al, 2013), showing variation of expression patterns in different experimental settings. For example, Oct4 was found to be required for fin regeneration in zebrafish (Christen et al, 2010), but inhibited lens regeneration in newts (Bhavsar and Tsonis, 2014).…”

Lens regeneration from the cornea requires suppression of Wnt/β-catenin signaling

“…This process is initiated when signals from the neural retina are able to reach the cornea epithelium, upon perforation of the cornea endothelium and removal of the lens (Freeman, 1963; Reeve and Wild, 1978). While cornea-lens regeneration has traditionally been described as transdifferentiation of the cornea, a different model has emerged suggesting that the regenerated lens may instead be derived from a population of basal stem cells or transient amplifying cells in the cornea which possess an oligopotent capacity to give rise to a new lens (Perry et al, 2013). However, the cellular signaling events needed to initiate lens regeneration in these cornea cells is not understood.…”