The optx2 homeobox gene is expressed in early precursors of the eye and activates retina-specific genes

Abstract

Vertebrate eye development begins at the gastrula stage, when a region known as the eye field acquires the capacity to generate retina and lens. Optx2, a homeobox gene of the sine oculis-Six family, is selectively expressed in this early eye field and later in the lens placode and optic vesicle. The distal and ventral portion of the optic vesicle are fated to become the retina and optic nerve, whereas the dorsal portion eventually loses its neural characteristics and activates the synthesis of melanin, forming the retinal pigment epithelium. Optx2 expression is turned off in the future pigment epithelium but remains expressed in the proliferating neuroblasts and differentiating cells of the neural retina. When an Optx2-expressing plasmid is transfected into embryonic or mature chicken pigment epithelial cells, these cells adopt a neuronal morphology and express markers characteristic of developing neural retina and photoreceptors. One explanation of these results is that Optx2 functions as a determinant of retinal precursors and that it has induced the transdifferentiation of pigment epithelium into retinal neurons and photoreceptors. We also have isolated optix, a Drosophila gene that is the closest insect homologue of Optx2 and Six3. Optix is expressed during early development of the fly head and eye primordia.

Figure 1

Comparison of Chicken Optx2 amino acid sequence with Mouse Optx2 and Six3. Top line: chicken Optx2 amino acid sequence. Line indicates homeodomain. Second line: mouse Optx2. Third line: mouse Six3. Dash: identity to chicken Optx2. Dots indicate absence of corresponding amino acid, such as in the N terminus of Six3.

Figure 2

Comparison of homeodomain sequences. Lines 1–8: Homeodomain amino acid sequence, Optx2, chicken and mouse. Comparison with Six3, mouse (29, 49); optix, Drosophila melanogaster; sine oculis, D. melanogaster (13, 14); Six1, mouse; Six2, mouse (41); Arec3/Six4, mouse (27; 49); and Pax6, mouse (6). Dash indicates identity to Optx2. Dot indicates absence of corresponding amino acid.

Figure 3

Expression of Optx2 during chicken development. Wholemount in situ hybridization of embryos and explants; 10 μm sections. Orientation: dorsal up (B–G). Blue/purple signal: Optx2 mRNA. (A Left) Gastrula (st 5), dorsal view, anterior up. Signal in eye field region. Arrow: Hensen’s node. (A Right) Neural plate embryo (st 7). Signal in prospective forebrain and head ectoderm. (B) Stage 5 embryo, sagittal section, anterior left. Signal in prechordal mesoderm and overlying ectoderm of eye field. nc, notochord; hn, Hensen’s node. (C) Neural fold embryo (st 7+) transverse section through forebrain. Signal in neural plate, head ectoderm, and prechordal plate (arrow). (D) Optic vesicle stage (st 11), ventral view. Signal in the ventral forebrain and optic vesicles, with none detected at more posterior levels of the head or body. (E) Optic vesicle, forebrain (st 12+), transverse section, midline on the right. Optx2 signal restricted to distal and ventral optic vesicle, ventral forebrain. Note signal in optic vesicle neurectoderm and overlying surface ectoderm (lens placode). (F) Optic cup stage embryo (st 16). Signal in branchial arch clefts is an artifact. (G) Stage 16, transverse section, eye faces left. Note absence of signal in outer layer of optic cup (RPE). Decreased signal in the central lens placode, in contrast to the future cornea and conjunctiva. Signal in ventral hypothalamus (vh) and overlying ectoderm. (H) E17 chicken neural retina. Cone outer segments visible on upper edge of specimen. Optx2 mRNA is detected in all cell layers, including photoreceptor inner segment. pr, photoreceptors; inl, inner nuclear layer; ip, inner plexiform layer; gc, ganglion cells. (Scale: A, white bar = 1,000 μm; B, C, E, and G white bar = 100 μm; D, white bar = 500 μm; E, white bar = 300 μm; and H, black bar = 50 μm).

Figure 4

Induction of neural retina markers in RPE; Expression and map position of the Drosophila optix gene. E7 RPE cells were transfected with 120 ng of cmvOptx2 and 40 ng of cmvGFP (A, B, and E–J) or with 160 ng of cmvGFP plasmid DNA alone (C and D). After 72 hr, cultures were stained with anti-visinin (rhodamine). Fluorescence detection with rhodamine-red (B, D, and G–J) or GFP-green (A, C, E, and F). A and B depict the same set of cells, as do C and D. The cell in E is visinin-negative. In F, the cell indicated by an arrow is also visinin-positive, whereas the other cell is visinin-negative. (G and H) Visinin-positive cells induced in E7 RPE, with the appearance of immature photoreceptors developing in culture. (I and J) Visinin and GFP-positive cells derived from post-hatch RPE. (Scale: bars in A and J = 50 μm for A–H and J; I = 100 μm.) Drosophila: In situ hybridization with optix probe was carried out with 0–12 hr Drosophila embryos. (K) Stage 5 (2.5 hr) blastoderm embryo, anterior to left, dorsal up, Nomarski optics. Circumferential band of optix expression, 93–85% egg length. (L) 11–12 (7 hr) embryo, before germ band contraction. Parasagittal optical section, orientation as before. Optix signal in dorsal, bilaterally paired structures (white arrow), the clypeolabrum (anterior end of embryo), and roof of the stomodeum. (M) In center, stage 13 embryo, view of dorso–anterior aspect, showing signal in paired dorsal sites and clypeolabrum. (M Lower) gastrulating embryo. Dorso–anterior expression of optix. (N) Map position of optix at 43F–44A and sine oculis, as determined by hybridization to polytene chromosomes. Bar indicates extent of YAC DYE02–19, which contains optix.