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We report here that misexpression of the transcription factor Pax6 in the vertebrate Xenopus laevis leads to the formation of differentiated ectopic eyes. Multiple molecular markers indicated the presence of mature lens fiber cells, ganglion cells, Müller cells, photoreceptors and retinal pigment epithelial cells in a spatial arrangement similar to that of endogenous eyes. Lineage tracing experiments showed that lens, retina and retinal pigment epithelium arose as a consequence of the cell-autonomous function of Pax6. These experiments also reveal that the cell autonomous activity of misexpressed Pax6 causes the ectopic expression of a number of genes including Rx, Otx2, Six3 and endogenous Pax6, each of which has been implicated in eye development. The formation of ectopic and endogenous eyes could be suppressed by coexpression of a dominant-negative form of Pax6. These data show that in vertebrates, as in the invertebrate Drosophila melanogaster, Pax6 is both necessary and sufficient to trigger the cascade of events required for eye formation.
Fig. 1. Pax6 misexpression results in eye-related phenotypes. See Tables 1 and 3 for
summary of results. Xenopus embryos were injected with 160 pg Pax6 RNA in one animal
pole blastomere at the 16-cell stage and fixed at stage 48. (A) Ectopic lenses (arrows) in the
absence of retinal tissue. (B) Hematoxylin and eosin stained section showing an isolated
ectopic lens adjacent to surface ectoderm. The lens fiber cell mass is surrounded by
hematoxylin stained nuclei of the lens epithelial layer. (C) b-crystallin immunolabeling of
the ectopic lens in B. (D) Proximal eye defect in right eye displaying an extension of RPE
(arrow) towards the midline. (E) Ectopic eye displaying a lens-like structure (small
arrowhead) in association with an optic cup-like structure (large arrowhead), extension of
RPE towards the midline (white arrowhead) and additional ectopic RPE-like structures
(arrows).
Fig. 2. Proximaleye defects in embryos misexpressing Pax6.
Xenopus embryos were injected with 160 pg Pax6 RNA in one
animal poleblastomere at the 16-cell stage and fixed at stage 48
(A,B) and stage 41 (C). (A) Extension of RPE in right eye toward the
midline. (B) Expansion of RPE and positioning of eye cup adjacent
to forebrain region (arrowhead). (C) RedGal labeling in lefteye of
embryo coinjected with 400 pg of Nuc-lacZ RNA shows an RPE
expansion adjacent to the forebrain region (arrowhead) and ectopic
RPE-like structure (arrow). The dotted white line indicates the
reduced distance from the midline to the distal boundary of the eye
on the affected side. (D) Section through embryo displaying
proximaleye phenotype in which the expanded RPE is adjacent to
the forebrain (arrow); the arrowhead indicates photoreceptor cells
adjacent to the brain region; (inset) rhodopsin staining in the eye
region of the same section in D showing rod photoreceptor cells
adjacent to the brain (arrowhead). (E) Uninjected embryo showing
the normal Six3 expression pattern in the late neurula (stage 20).
(F) Pax6-injected embryo showing an expansion of Six3 expression
towards the midline (arrowhead) compared with that of E
(arrowhead) and diminished Six3 expression distally (asterisk).
Vertical dotted line indicates the midline, and the horizontal line the
dorsal limit of Six3 expression.
Fig. 3. Ectopic eyes induced by Pax6 misexpression
resemble normal eyes morphologically and histologically.
Xenopus embryos were injected with 160 pg of Pax6 RNA
in one animal pole blastomere at the 16-cell stage and
fixed at stage 48. (A-C,E,F) Ectopic eyes from different
embryos displaying eye cup (white arrowhead) and lens
(black arrowhead). (A) Ventral view; anterior, top.
(B,F) Side view; dorsal, top; anterior, right. (C) Dorsal
view; anterior, left. (E) Dorsal view; anterior, right; RPElike
extension from eye cup (C,E, arrow). (D) Dorsal view
of C showing that this particular ectopic eye is positioned
posterior to the otic vesicle (OV). (G-I) Hematoxylin and
eosin staining of coronal sections through (G) normal eye,
and (H,I) ectopic eyes. Section in H taken from ectopic
eye in E; section in I taken from ectopic eye in F. In I note
reverse orientation of lens (L) which is separated from the
epidermis (bottom) by ectopic retina and therefore not
visible in F. P, retinal pigmented epithelial layer; O, outer
nuclear layer; I, inner nuclear layer; G, ganglion cell
layer; L, lens; arrows indicate ciliary margin zone in
normal eye and region with similar morphology in ectopic
eyes.
Fig. 4. Ectopic eyes express markers of differentiated retinal cell
types and lens. Sections of endogenous normal eye (A,B,E), and
ectopic eyes (CD,F-H). DIC illumination is shown in A, C and G and
fluorescence illumination in B,D-F and H. Wild-type (A,B) and
ectopic (C,D,G,H) eyes contain lenses or lens-like structures
(C,D,G,H, arrowheads) which show immunoreactivity with antibodies
to b-crystallin (yellow labeling). The presence of photoreceptors is
demonstrated with rhodopsin immunoreactivity (green labeling) in
normal (B,E) and ectopic (D,F,H) eyes. The location of ganglion and
amacrine cells in the inner layers of the retina is indicated by labeling
with an antibody to Islet-1 (red nuclear labeling) in both normal (B
and E) and ectopic (C, D and F) eyes. Müller cells, indicated by blue
labeling (B,D-F) or green labeling (C) for glutamine synthetase, are
closely associated with islet-1-positive ganglion and amacrine cells in
the inner retinal layers of both normal (B,E) and ectopic (D,F) eyes.
High magnification section through retina in examples of endogenous
(E) and ectopic (F) eyes indicate that retinal lamination follows the
expected pattern. The location of the pigmented cell layer in (E,F) is
indicated by the dashed lines. In all cases, ectopic eyes were
associated with a layer of pigmented RPE-like cells (C,G, arrows).
The inset in H shows higher magnification of rhodopsin stained cells
of ectopic eyes with a similar morphology to that of endogenous rods.
Fig. 5. Ectopic eye formation by Pax6 is autonomous. Embryos were
coinjected with 160 pg Pax6 and 400 pg of Nuc-lacZ RNA and
stained with the RedGal substrate. (A,B) Embryos displaying ectopic
eyes in whole-mount (black arrows); lens is indicated by white arrow
in B. (C) Section through ectopic eye of embryo in B showing
RedGal labeling in the eye cup and surrounding RPE (arrows);
dotted line in C and D indicates boundary of RedGal labeling.
(D) Immunofluorescence labeling of section shown in C; XAR (RPE;
purple), rhodopsin (rod photoreceptors; green) and Islet-1 (ganglion
cells; red). (E,F) Adjacent section through the ectopic eye in C and D
showing (E) RedGal labeling in lens epithelium surrounding lens
fiber cells and (F) b-crystallin labeling; arrowheads in E and F
indicate boundary of the lens.
Fig. 6. Ectopic gene expression in embryos misexpressing Pax6. See
Table 2 for summary of results. In situ hybridization was performed
on embryos injected with 160 pg Pax6 and 400 pg Nuc-lacZ RNAs.
Embryos at neurula stages showing ectopic expression (blue labeling
and arrowheads) of (A) Rx (embryo tilted to show dorsal aspect), (B)
Otx2, (C) Six3 and (D) endogenous Pax6. (E-G) Embryos at the early
neurula displaying ectopic Rx expression (arrowheads). (G) Ectopic
Rx staining (arrowhead) localized within a region of RedGal labeling
(red).
Fig. 7. Inhibition of eye formation in whole embryos and animal
caps by a truncated version of Pax6. (A) Embryos were injected with
60 pg Pax6DCT RNA in one dorsal animal blastomere at the 8-cell
stage and fixed at stage 45. Embryos displayed phenotypes ranging
from a reduction in eye size (left panel) to the complete loss of eye
structures (middle panel). (B) Embryos were injected in both
blastomeres of 2-cell embryos with a fixed amount of RNA for the
neuralizing factor follistatin (1 ng) and challenged with increasing
levels of Pax6DCT (lanes 1-5 show 6, 12, 25, 50, 100 pg of Pax6DCT
RNA respectively). Animal cap ectoderm was explanted at midblastula
(stage 8.5) and processed for RT-PCR at stages 20 and 41 as
indicated on the left side of the panel. An elongation factor-1a (EF-
1a) cDNA product was amplified as an internal loading control for
animal caps at both stages. Lane 6: uninjected animal cap control.
Lane 7: embryo minus-reverse transcriptase negative control. Lane 8:
whole embryo positive control. NCAM, neural cell adhesion
molecule.
