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Figure 1. EFTFs induce an eye field-like transcriptional profile.
(A) Location of the eye field (EF), PNP, and LE tissue isolated from stage 15 Xenopus embryos for microarray analysis. (B) Venn diagram identifying the number of genes induced 2-fold or greater in EFTF-expressing pluripotent cells (EFTF-PC, blue) and enriched 2-fold or greater in eye field (red), PNP (orange), or LE (yellow). (C) Relative expression levels of transcripts required for eye formation (boxed in pink; also see Table S1). Known markers of PNP (boxed in orange) and LE (boxed in yellow) are included for comparison (also see Table S2). Microarray data sets for pluripotent cells (PC), EFTF-PC, EF, PNP, LE, and whole embryos (WE) are shown. High and low expression is indicated in red and blue, respectively. The probe sets detecting these transcripts do not hybridize to the injected RNA. Consequently, the EFTF induction detected here is transcription from the endogenous genes (pax6, rx/rax, six3, and otx2; noted by asterisk).
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Figure 2. EFTF-expressing pluripotent cells form eye-like structures in tadpole flank.
(A) Schematic illustrating the expected retinal layers of the eye including the outer nuclear (ONL), inner nuclear (INL), and ganglion cell (GCL) layers. The mitotically active CMZ is indicated in red. (B) Transverse section of a stage 41 wild-type retina. Dashed lines mark the location of the plexiform layers. The eye is oriented so that dorsal is to the left. (C) Flank eyes expressed YFP throughout, contained an RPE and a trilayered morphology with putative inner and outer plexiform layers (dashed lines in D). Markers were used to detect retinal cell classes. (C) Whole tadpole bright field image overlayed with fluorescent image, showing flank eye (yellow box) on top of gut (dashed line). Inlay is a magnified view of the boxed flank eye. (D) Cryostat section (12 ) shows cells labeled for the retinal ganglion cell (RGC) marker, hermes, by in situ hybridization. (E) R5 antibody labels Mler glia (M red), which extend processes the breadth of the flank retina. (F) GABA labels horizontal and amacrine cells (HC, AM; red) of the INL. (G) Cells expressing YFP (green) co-express the cone photoreceptor (cPR) marker Calbindin (yellow). (H) Cells expressing the rod photoreceptor marker XAP2 (rPR; red); BrdU-immunoreactivity (yellow) identifies mitotically active cells in the periphery of the same flank retina. Scale bars, 25 .
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Figure 3. Schematic of the transplant procedure used to assess the fate of EFTF-expressing pluripotent cells.
(A) YFP-only, or (B and C) EFTF RNAs were injected into both blastomeres of two-cell stage embryos from CAG-Venus YFP transgenic X. laevis. At stage 9, pluripotent cells were removed and cultured to the equivalent of stage 15. For experiments requiring complete eye field replacement (A and B), one half of the eye field was surgically removed from wild-type, stage 15 (host) embryos, and replaced with the donor cells from one half of an explant. (C) To create mosaic eyes, approximately one-half of one eye field was removed and a size-matched fragment of donor tissue was grafted to the host eye field. The location of the eye field is shaded red. Schematic embryos adapted from Nieuwkoop and Faber and Eagleson and Harris [38],[42].
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Figure 4. EFTF-expressing pluripotent cells form morphologically normal eyes.
(A) Control YFP-expressing pluripotent cells (YFP) form only epidermis when grafted to embryos after removal of the endogenous (left) eye field. Bright field and fluorescent images show the location of transplanted cells. (D) In contrast, EFTF-expressing pluripotent cells (EFTF/YFP) generate an eye. YFP fluorescence demonstrates the eye originates from donor tissue. Induced eyes grow throughout the life of the animal (E, H), and form the characteristic trilayered structure of a normal retina (G). (E) Retinal ganglion cell axons exit the eye as the optic nerve (white arrow), pass under the brain and out of view then reappear on the contralateral side (arrowhead). Donor cells are sometimes also found in forebrain (yellow arrow) and epidermis (red arrow). (F) YFP expression is detected throughout the eye, in RGC axons (arrowhead) and forebrain (arrow). (I and J) Eye fields (EF/YFP) also generate eyes. In addition to the eye, epidermis, and forebrain, some musculature and olfactory tissue are also derived from donor eye field. White arrow points to ganglion cell axons, while red arrow points to ocular muscle. Arrowhead indicates the location of YFP-positive retinal axons that have projected to the brain. (A, I, and J) stage 45 tadpoles; (F and G) stage 46 tadpole; (H) 3-mo-old froglet. In (G), a sectioning artifact introduced a few YFP-negative epidermal cells between the optic cup and lens (Le). Scale bars, 100 .
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Figure 5. EFTF-expressing pluripotent cells generate all retinal cell classes, RPE, and mitotic cells in the CMZ.
(A) Control, pluripotent cells (green) never formed retinal tissue. (B) In contrast, EFTF/YFP-expressing cells generate mosaic retinas with columns of cells spanning the entire retinal width. Retinal ganglion cell axons (arrowhead), RPE (arrow), and rod photoreceptors (stained red for XAP2) are donor derived. (C) Bright field image of a representative retina to illustrate the expected location and morphology of retinal cell classes. The color of cells in (C) is matched to the appropriate cell in (D). (D) Examples of single YFP-labeled cells identified on the basis of their distinctive morphology (outlined), location within the mosaic retina, and expression of cell class specific markers. Cells are oriented with the RPE side of the retina at the top and lens to the bottom. Z-series images were used to determine the physical boundary of each cell (see Figures S4, S5, S6 for examples). Cones (D) and rods (E) appear greenish/yellow when costained for YFP and Calbindin (red) or XAP2 (red), respectively. XAP2 labels rod outer segments strongly, while YFP is predominantly localized to rod inner segment. Muller glial (F) and horizontal (G) cells express YFP. Bipolar (H), amacrine (I), and retinal ganglion (J) cells double labeled for YFP and Calretinin appear reddish-yellow. (K) Expression pattern of cell class markers is continuous through host and induced regions of mosaic retinas (see also Figure S2). Mosaic retinas stained for Calbindin (cones, K), R5 (Mler glia, L), GABA (horizontal and amacrine, M), and Islet-1 (retinal ganglion and inner nuclear layer cells, N). Host (YFP) cells appear red when stained for cell class specific markers, while double-labeled, donor-derived cells (arrows, arrowheads, and carrots) appear yellowish-red. (O) Comparison of RGC, inner nuclear layer, and rod photoreceptor cell densities in endogenous and induced regions of mosaic retinas. Graphs show meanEM, n>500 cells for each cell class. (P and Q) BrdU-labeled (red) cells detected in the CMZ (outlined in Q) of induced eyes. Sections in (A, B, P, and Q), (C), and (K) are of stage 46, 50, and 54 tadpoles, respectively. Scale bars, (A) = 50 ; (D) = 10 ; (P, Q) = 25 .
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Figure 6. Eyes generated from induced retinal cells are functional.
(A and B) ERG responses of control (WT) and induced eyes (IE) are similar. (A) Representative ERG response of the induced and control eye recorded from the same animal. Traces represent the average of eight or more responses. Flash was 520 nm, 20 ms in duration. Light intensity is indicated. (B) The b-wave magnitudes of two induced eyes (IE1, IE2) increase with flash intensity. Open symbols represent the average of eight control animals. Plot shows meantandard deviation. Averages are fit with a Michaelis-Menten function. (C and D) Background color preference histograms of control (C) and experimental (D) animals. Tadpoles were tested before (unoperated, U), and following single (SA) and double (DA) retinal axotomy. White and black histogram bars indicate average percent time spent on white or black side of test tank. Graphs show meantandard error of the mean. n = number of 2-min trials. (E and F) Sections of the control (E) and induced (F) eyes of the animal tested in (D). Cell nuclei, rod outer segments, and donor-derived retina are blue (DAPI), red (XAP2), and green (Venus YFP), respectively. Scale bar, 100 .
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Figure 7. Noggin-treated pluripotent cells generate retinal cells in vitro and form functional eyes in vivo.
(A) Retinal cells formed in cultured explants treated with Noggin protein. (A) Rosettes (arrowheads) and pseudo outer nuclear layers (arrow) are detected in explants triple stained for nuclei (blue; DAPI), cone (green; Calbindin), and rod photoreceptors (red; XAP2). (B) Cultured cells not only expressed photoreceptor specific markers, but also had morphologies typical of immature photoreceptors including small outer segments and oil droplets (arrow). (C) A Calretinin-expressing cell (red) shows characteristic bipolar cell morphology, extending processes toward a nearby layer of photoreceptors. Arrows indicate the location of representative oil droplets in the photoreceptor layer. (D) Pluripotent cells from YFP transgenics form eyes when treated with Noggin protein and grafted to the eye field of stage 15 wild-type hosts (these stage 32 animals were photographed 1 d posttransplant). (E) The background color preference assay demonstrates that Noggin-induced retinas are functional. No statistical difference was observed prior to (U) and following single axotomy (SA) to blind the control eye. However, following axotomy of the RGC axons from the induced eye (DA), animals showed no background color preference. Graphs show meantandard error of the mean. * p = 0.002 (E). n = number of 2-min trials. (F) Eye generated using Noggin-treated pluripotent cells (tested in E) was completely donor derived. YFP expression is detected in axons projecting from the induced eye (G, arrow) to the brain (G, arrowhead). YFP expressing cells are also observed in the surface ectoderm and left half of the forebrain (G). (H) Noggin-induced eye stained for YFP (green) throughout demonstrating it was completely donor derived. Induced eye was also stained to detect rod photoreceptors (XAP2, red) and nuclei (DAPI, blue). rPR, rod; cPR, cone photoreceptor; BP, bipolar cell.
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Figure S1. An unsupervised hierarchical clustering algorithm groups the transcriptional profile of eye field and EFTF-expressing pluripotent cells together.
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Figure S2.
EFTF-expressing pluripotent cells differentiate as retinal cells and incorporate seamlessly with host cells to form mosaic retinas.
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Figure S3.
Example demonstrating how individual cell classes were identified based on morphology and cell class-specific markers.
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Photoreceptors Ab1 expression (red channel) in NF stage 48 retina.
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Muller Glia Ab1 expression in NF stage 41 Muller Glia.
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Calb1 Ab1 staining in cone photoreceptors in sectioned Xenopus laevis eye
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Gaba Ab1 staining in sectioned Xenopus laevis eyes.
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Isl1 Ab2 staining of sectioned NF stage 41 Xenopus laevis eye
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Calb2 Ab1 staining of sectioned NF stage 41 Xenopus laevis eye
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