Chow RL et al. (1999), Pax6 induces ectopic eyes in a vertebrate.

XB-ART-12377

Development 1999 Oct 01;12619:4213-22. doi: 10.1242/dev.126.19.4213.

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Pax6 induces ectopic eyes in a vertebrate.

Chow RL , Altmann CR , Lang RA , Hemmati-Brivanlou A .

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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.

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Species referenced: Xenopus laevis
Genes referenced: dnai1 fst glul isl1 ncam1 nucb1 otx2 pax6 rho rpe six3

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	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. Proximal eye defects in embryos misexpressing Pax6. 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,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 left eye 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 proximal eye 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.