Perry KJ et al. (2013), Expression of pluripotency factors in larval ep...

XB-ART-46433

Dev Biol 2013 Feb 15;3742:281-94. doi: 10.1016/j.ydbio.2012.12.005.

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

Perry KJ , Thomas AG , Henry JJ .

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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 h 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., one involving cellular dedifferentiation followed by redifferentiation). Our combined observations provide evidence that a population of stem cells exists within the Xenopus cornea. We hypothesize that the basal epithelium contains oligopotent epithelial stem cells that also represent the source of regenerated lenses in the frog. Future studies will be required to clearly identify the source of these lenses.

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Species referenced: Xenopus laevis
Genes referenced: dnai1 dyrk1a eef1a1 klf4 myc odc1 pou5f3 pou5f3.3 sox2 tac1 tp63 wnt3a wnt5a zic2 zic5

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	Fig. 1. Morphology of larval cornea tissues in whole mount and transverse sections of Xenopus laevis. (A) Confocal images of an intact excised cornea (pelt) of st502 larvae. (A) Outer epithelial layer labeled with Phalloidin (green), DAPI labeled nuclei (blue) and merged images, respectively. (D) Basal epithelial layer labeled with Phalloidin (green), DAPI (blue) and merged images, respectively. (G) Deeper fibrillar layer labeled with Phalloidin (green), DAPI (blue) and merged images, respectively. (J) DIC image of transverse section of control larval eye showing thin, dual-layered epithelium. (K) Corresponding Hoechst nuclear stained image to that seen in J showing flattened nuclei. (L) DIC image of transverse section of 5-day regenerating larval eye with thickened cells of the epithelium. (M) Corresponding Hoechst nuclear stained image to that seen in L. Gray arrowhead points to one nucleus of the outer cornea epithelial layer and white arrowhead identifies a nucleus in the basal epithelial layer. The gray arrowhead points to the nucleus of a cell of the outer epithelial layer and one located in the basal cornea epithelium is denoted with a white arrowhead. ic; inner cornea; ln, lens; nr, neural retina; oc, outer cornea. Scale bar in M equals 6 μm for A, and 100 μm in J.
	Fig. 2. (A) Larval (st48) Xenopus specimens showing distribution of acetylated tubulin labeled nerves in the head. (A) Right lateral view of the head with anterior to the right, and dorsal to the top of the figure. (B) Superior, dorsal view of the right side of the head with anterior to the left. Asterisks mark branches of sensory nerves that serve the cornea epithelium in A or their origins in B. (C) Confocal images showing different depths of acetylated tubulin immunostaining (red), cell boundaries labeled with phalloidin (green) and DAPI nuclear stain (blue). (C) Acetylated tubulin stained nerves of the outer epithelial layer. (D) Basal epithelial distribution of acetylated tubulin labeled nerves. (E) Fibrillar layer containing labeled nerve trunks with branching structures. (F) Confocal image showing distribution of acetylated tubulin labeled nerves in the cornea (red). (G) Corresponding image showing distribution of Substance P (green). (H) Merged images from F and G showing overlapping acetylated tubulin and Substance P protein localization. an, abducens nerve (CN VI); fb, forebrain; fn, facial nerve (CN VII); gn, glossopharyngeal nerve (CN IX); ion, infraorbital nerve; mn, mandibular nerve; mnb, fine branches of the mandibular nerve; ncn, nasociliary nerve (with m and l, labeling its terminal medial and lateral branches, respectively, which pass to the sides of the olfactory organs (nose)); ofn, olfactory nerve (CN I); omn, oculomotor nerve (CN III); on, optic nerve (CN II); opn, ophthalmic nerve; rn, retrograde branch of nasociliary nerves; son, supraorbital nerve; tn, trochlear nerve (CN IV); tgn, trigeminal nerve (CN V). Scale bar in H equals 150 μm in A and 6 μm in C.
	Fig. 3. Quantitative PCR (qPCR) results for sox2, p63 and oct60 expression during lens regeneration in stage 502 Xenopus laevis larval cornea epithelia. Relative expression levels were compared to control cornea epithelial expression and normalized against odc expression at the same larval stages (st502). Data for each time point was run in triplicate and represents independently pooled cornea samples collected from 0 h (control unoperated corneas), 4 h, 12-h, 1-day, 3-day and 5-days after lens removal. Error bars are indicated in black. Significant expression changes as compared to control unoperated corneas are denoted with an asterisk at the top of each bar graph. Significant expression changes between various key time points compared are noted underneath the X-axis. * denotes p values<0.05 using Students t-test.
	Fig. 4. Antibody labeling of sox2 (red) in whole Xenopus larval cornea epithelium (st502). (A) Antibody labeling in whole corneas showing the distribution and appearance of labeled cells in regenerating corneas. (A) Control cornea with very few sox2 labeled cells located around the periphery in the limbal zone. (B) Regenerating cornea just 4 h after lens removal. Note the increased number of labeled cells that have appeared in the central cornea. (C) Regenerating cornea 5 days following lens removal with a decreased number of sox2 labeled cells, which are again restricted to the periphery. (D) Representative higher magnification views showing sox2 protein labeling in irregularly-shaped cells of the cornea. The outer periphery or limbal zone is located to the left of each photo and the center of the cornea is towards the right. (D) Distribution of sox2 antibody (red). Some low level sox2 expression is also detected throughout other cells of the cornea epithelium. (E) Corresponding view showing distribution of DAPI stained nuclei (blue). (F) Merged images from D and E. (G) Confocal images of a 3-day regenerating cornea, showing one irregular sox2 expressing cell located in the basal layer (red). Cell boundaries and fibrillar layer are counterstained with phalloidin (green) and DAPI nuclear stain (blue). (G) Outer cornea epithelium. (H) Basal cornea epithelium. The sox2 expressing cell appears to contain multiple nuclei or multi-lobed nucleus. (I) Fibrillar layer with no apparent sox2 labeling. (J) Confocal images of 1-day regenerating cornea epithelium with sox2 antibody labeling (red). (J) Outer cornea epithelium. (K) Basal cornea epithelium with diffuse sox2 label throughout the cells. (L) Deeper fibrillar layer with a multinucleated cell brightly labeled with sox2. This cell appears to either be dividing or squeezing through two adjacent unlabeled cells. cc, central cornea; lz, limbal zone. Scale bar in L indicates 220 μm for A, 40 μm for D, 6 μm for G, and 8 μm for J.
	Fig. 5. Fluorescence light microscopic and confocal images showing antibody labeling of Xenopus whole cornea epithelium (st502). (A) p63 (ΔNp63 isoform) protein labeling of the cornea epithelium. (A) p63 labeling is represented in red. (B) Corresponding image to A with DAPI labeled nuclei (blue). (C) Merged images from A and B. (D) Confocal imaging of p63 antibody labeling. (D) Outer epithelium with no p63 labeling. Cell boundaries and fibrils are counterstained with phalloidin (green). (E) Localized p63 labeling (purple) in every nucleus of the basal epithelium. (F) Fibrillar layer with no p63 labeling. (G) c-myc protein labeling in the peripheral region of the cornea (limbal zone). (G) c-myc localization to multi-lobed cells (red). (H) Corresponding image to G with DAPI stained nuclei. (I) Merged image of G and H. (J) Confocal images of c-myc labeling in cells of the cornea epithelium (limbal zone). (J) Outer cornea epithelium with projections of c-myc labeled cells. (K). Basal cornea epithelium displaying irregular shaped c-myc expressing cells. (L) Deeper fibrillar layer displaying no c-myc labeled cells. Scale bar in L indicates 25 μm for A, 6 μm for D, 50 μm for G, and 10 μm for J.
	Fig. 6. EdU labeling of proliferating cells in isolated intact control Xenopus larval cornea epithelia (pelts) following a single pulse of EdU labeling. Animals were fixed at various time points following injection and EdU detected with fluorescent azide 594. White arrowheads point to the location of the central attachment point of the cornea epithelium to the cornea endothelium. (A) Cornea 1-day post-EdU injection (st502). (A) Fluorescent EdU labeled nuclei (red). Representative pairs of EdU labeled nuclei are denoted with gray arrowheads, of which many can be seen in this specimen. (B) EdU labeled image from A merged with Hoechst 33342 labeled nuclei (blue). (C) Cornea 5-days post-EdU injection (st52). (C) Fluorescent EdU labeled nuclei. Representative clusters of EdU labeled nuclei are noted with gray arrowheads. (D) Merged images from C and corresponding Hoechst labeled nuclei (blue). (E) Cornea 14-days post-EdU injection (st545). (E) Fluorescent EdU labeled nuclei. Representative clusters of EdU labeled nuclei are pointed out with gray arrowheads. (F) Merged images from E and corresponding Hoechst 33342 labeled nuclei. (G) Cornea 28-days post-EdU injection (st567). (G) A few fluorescent EdU labeled nuclei are present in this cornea (denoted by gray arrowheads). (H) Merged images from G and corresponding Hoechst 33342 labeled nuclei. (I) Sections of eyes from animals injected with EdU 14 days prior to fixation. (I) Section of 14-day control case showing the central cornea overlying the optic cup where EdU label occurs mostly in nuclei of the basal epithelium. (J) Corresponding image to I showing additional location of all nuclei labeled with Hoecht. (K) Section of 14-day EdU labeled case (same case shown in K) showing the periphery of the cornea. (K) EdU labeled cells from 14-day control case are shown in red and merged with Hoechst labeled nuclei in L. Scale bar in L indicates 100 μm.
	Fig. 7. EdU labeling (red) of nuclei in proliferating cells during different time point of lens regeneration in Xenopus eyes. (A) Cornea 1-day post EdU injection and lens removal (st502). (A) Fluorescence light micrograph of cornea pelt showing presence of EdU labeled nuclei (red). (B) Corresponding image to A showing additional location of all nuclei labeled with Hoecht (blue). (C) Higher magnification confocal image of 1-day regenerating cornea showing outer cornea epithelial layer with no appreciable EdU labeling. Nuclei are stained with Hoechst. (D) Deeper focal plane showing corresponding basal cornea epithelial layer with presence of EdU labeled nuclei. (E) Deeper fibrilar layer showing presence of some nuclei, one of which is EdU labeled. (F) Regenerating cornea 5-days post-EdU injection and lens removal (st52). (F) Fluorescent EdU labeled nuclei in 5-day regenerating cornea. Gray arrow shows location of intense EdU labeling. (G) Corresponding image to F showing additional location of all nuclei labeled with Hoecht (blue). The location of a regenerating lens placode is enlarged in the inset. This could represent the point of attachment of a larger lens vesicle that detached from the cornea upon excision from the animal. (H) Higher magnification confocal image of an organized regenerating lens placode 5-days after EdU addition and lens removal. (H) Image showing lens placode with EdU labeling in red. (I) Corresponding image of H showing nuclei labeled with Hoecsht. (J) Merged fluorescence images in H and I. (K) Section of eye showing EdU labeled cells present in a regenerated lens 14 days after EdU injection and lens removal. The cornea is at the upper edge of these images. (K) Section of eye showing the lens containing red EdU labeled nuclei. (N) Merged image from K combined with corresponding Hoechst labeled nuclei. (M) Cornea implantation experiment where larvae (st502) were labeled with EdU, chased for 7 days, following which the outer cornea was excised and implanted into the vitreous chamber of an unlabeled control larvae (st502). (M) DIC image of eye 7-days after cornea implant. Small regenerating lens is outlined in black. (N) Corresponding image to M showing EdU-labeled nuclei. Note EdU labeled nuclei within the regenerating lens, outlined in white. (O) Merged fluorescence images shown in M and N. imc, EdU labeled implanted donor cornea; ln, lens; nr, neural retina; oc, outer cornea; pce, peripheral cornea epithelium; vc, vitreous chamber. Scale bar in T equals 100 μm in A, F; 60 μm in G (inset); 6 μm in C; 10 μm in H; 15 μm in K, and 40 μm in M.
	Supplementary Fig. S1. Expression of pluripotency genes in control corneas (C), during days 1 of lens regeneration (R), and in flank (F) epithelium as assayed by RT-PCR. All PCR products were verified by sequence analysis. 1 kb standards (st) are to the far left, as indicated.

References [+] :

Arresta, Lens-forming competence in the epidermis of Xenopus laevis during development. 2005, Pubmed, Xenbase