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Fig.1. Ectopic eye formed
in vitro shows similar morphology
to a normal eye. (A) Schematic
diagrams showing the formation
of the eye. Dorsal lip and lateral
marginal zone (LMZ) cells from
early gastrulae were sandwiched
between two sheets of animal
cap from late blastulae. (B,D)
External view of the normal
embryo (B) and the explant (D).
Lens and retinal-pigmented epi-
thelium (RPE) could be observed
(D, arrowhead). (C,E) Hematoxy-
lin and eosin staining of a coronal
section through the normal
embryo (C) and the explant
(E). The eye showed similar
morphologic characteristics to
the normal eye. NR, neuroretina;
RPE, retinal pigmented epithe-
lium. Scale bar indicates 1 mm
(B,D) or 50 μm (C,E). (F) Reverse
transcription-polymerase chain
reaction (RT–PCR) analysis of
the explants was performed at
stage 42. Note that expression of
the mature lens marker, ô°-crys-
tallin was detected only when
both dorsal lip and LMZ cells
were sandwiched between the
two sheets of animal cap (lane 5).
When dorsal lip alone was com-
bined with animal caps, which
was the condition that resulted in
induction of the most anterior
regions, the mature lens marker
was not detected (lane 4). Non-
treated animal cap, dorsal lip,
and LMZ cells combined with
animal caps did not express any
markers (lanes 1, 2, 3). otx-2,
forebrain marker; HoxB9, spinal
cord marker; NCAM (neural cell
adhesion molecule), pan-neural
marker. Detection of ornithine
decarboxylase (ODC) acts as
an internal control. ODC RT–
indicates experiments without
reverse transcriptase. ac, animal
cap; DL, dorsal lip; LMZ, lateral
marginal zone cells; WE, whole
embryo. (G) Bar graph summarizes the results from the observation of tissue differentiation. Neuroretina, RPE, and lens were observed frequently in the sandwich-explants combining the dorsal lip and LMZ cells.
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Fig 2. Mature, differentiated cells of the retina and lens were observed in the eye induced in vitro. Electron microscopy of the in vitro eye. Mature lens (A) and photoreceptor cells (B) are present in the eye induced in vitro. d, disc membrane; i, inner segment; n, nucleus; o, outer segment.
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Fig. 3. The eye induced in vitro was derived from animal cap cells. (A,B) Lineage labeling in dorsal lip cells. Explant, external view (A) and histologic section (B). TRDA signal (red fluores- cence) was observed in the notochord only (B, white arrow). (C,D) Lineage labeling in animal cap cells. Explant, external view (C) and histologic section (D). TRDA signal was observed in the eye only (D, white arrow), indi- cating that the eye formed in vitro was derived from animal cap cells. The white line in A and B indicate the approximate position of the section. Scale bar indicates 1 mm (A) or 10 μm (B).
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Fig. 4. The eye formed in vitro was transplanted into stage33 tadpoles. (A) Schematic dia- grams of lineage labeling (a) and transplantation (b). The 2-cell stage embryos were injected with 8 nL of 0.5% Texas red-dextran- amine (TRDA) at the animal pole. Using these embryos, the eye was induced and cultured until early tadpole stage (stage 33) (a). The left eye was surgically removed from the recipient tadpole (stage 33), and the eye formed in vitro was extirpated from the explant and transplanted into the tadpole (b). (B) External views of the recipient stage 33 tadpole (top) and the donor explant (bottom). Black arrow indicates eye induced in vitro. (C) Magnified view of B. TRDA signal (red fluorescence) was observed in the explant only. (D,E) Tadpole showing the trans- planted in vitro eye. (D) The graft was inserted at the position of the original eye. (E) Red fluorescence could be observed at the grafted eye. White arrowhead indicates the grafted eye. Scale bar indi- cates 1 mm (B,C,E).
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Fig. 5. The grafted eye formed in vitro connected to the recipient and this eye was retained after metamorphosis. (A) Dorsal view of the recipient 15days post- transplantation. The grafted eye was observed at the left side of the recipient (white arrowhead). (B) Lateral view of the recipient. RPE and lens could be seen. (C) High magnification view of A showing TRDA signal (red fluor- escence) on the grafted eye. Note that the optic nerve extended from the grafted eye; the white arrow indicates TRDA signal corresponding to the extending optic nerve. on, optic nerve; tn, trigeminal nerve. (D) TRDA- labeled animal caps were trans- planted into the tadpoles at stage 33 as a negative control for the transplantation procedure. Eye structures were not induced in these grafted tissues (white arrowhead). (E,F) Electron micro- scopy of the optic nerve of normal eye (E) and the transplanted eye (F). The optic nerve extending from grafted eye had the same structure as a normal optic nerve. son, sheath of optic nerve; ra, retinal axon. (G) External view of a juvenile frog. After metamorph- osis, the grafted eye bonded with the host frog. Black arrowhead indicates the transplanted eye (left side of the frog), suggesting that the grafted eye connected to the host tadpole after meta- morphosis. Scale bar indicates 1 mm (A-D).
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Fig.6. The regenerated optic
nerve reached the tectum of the
host tadpole. (A) Dorsal view of
the recipient at 15days post-
transplantation. The grafted eye
was seen at the left side of the
recipient (white arrowhead). The
optic nerve extending from the
grafted eye (white arrow) was
labeled with the crystal of DiI
(black arrow). (B) Lateral view of
DiI-labeled tadpole. The DiI
signal (red fluorescence) could
be observed at the optic nerve,
and the labeled optic nerve reached the tectum of the host brain. fb, forebrain; hb, hindbrain; ot, optic tectum; th, thalamic visual center; bon, basal optic nucleus. Scale bar indicates 1 mm (A, B).
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Fig. 7. The eye induced In vitro shows normal function. (A,B) Dorsal view of stage 50 tadpoles. The tadpole with both eyes removed at stage 33 (A) and the transplanted TRDA-labeled eye induced in vitro (B). (C,D) External view of a juvenile frog. (C) The control eyeless frog was a dark color. (D) The frog with the grafted eye formed in vitro was a light color. These results suggest that the frog with the grafted eye formed in vitro could sense the brightness of its surroundings using the transplanted eye and was thereby able to control its skin color.
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