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Khosrowshahian F
,
Wolanski M
,
Chang WY
,
Fujiki K
,
Jacobs L
,
Crawford MJ
.
???displayArticle.abstract??? Pitx3 is expressed in tissues fated to contribute to eye development, namely, neurula stageectoderm and pre-chordal mesoderm, then presumptive lensectoderm, placode, and finally lens. Pitx3 overexpression alters lens, optic cup, optic nerve, and diencephalon development. Many of the induced anomalies are attributable to midline deficits; however, as assessed by molecular markers, ectopic Pitx3 appears to temporarily enlarge the lens field. These changes are usually insufficient to generate either ectopic lenses to enlarge the eye that eventually differentiates. Conversely, use of a repressor chimera or of antisense morpholinos alters early expression of marker genes, and later inhibits lens development, thereby abrogating retinal induction. Reciprocal grafting experiments using wild-type and morpholino-treated tissues demonstrate that Pitx3 expression in the presumptive lensectoderm is required for lens formation. Contradictory to recent assertions that retina can form in the absence of a lens, the expression of Pitx3 in the presumptive lensectoderm is critical for retina development.
Figure 2. Spatial expression pattern of xPitx3 assayed by whole-mount in situ hybridization. A-C: Early stage embryos seen laterally from a slightly anterior perspective (right) in (A), head on (B), and from the right side with the head twisted to face out on the right (C). D: Anterior top view of a stage 30 embryo showing that xPitx3 is highly expressed in the differentiating lens and Rathke's pouch (the cement gland is at the right). E,F: A lateral view (E) and a ventral view (F) of a tadpole at stage 39. xPitx3 is expressed throughout heart and gut development. G-I: Sense control embryos (G) reveal no hybridization of probe, whereas both gut and heart display asymmetrical labeling during looping of both organs (H,I). BR, branchial arch; EA, eye anlage; J, jaw musculature; L, lens; OV, otic vesicle; R, Rathke's pouch; S, somite; S/H, stomodeal-hypophyseal anlage; h, heart.
Figure 3. A-E: Parasagittal section (A) and anterior transversal sections (B-E) of whole-mount in situ hybridization of xPitx3 during the pituitary and eye development. A: Anterior is oriented right, and the arrowhead indicates the prechordal mesoderm and eye anlage region. R, the pituitaryprimordium-Rathke's pouch; LE, lensectoderm; LP, lens placode; L, lens.
Figure 4. B-F: Dorsal view of tadpoles injected with xPitx3 at the one-cell stage (B-E), and xPix3/engrailed repressor chimeric RNA at the two-cell stage embryos (F). Overexpression of xPitx3 induced severe eye defects. A: Uninjected control tadpole at stage 46. B: Anteriorhead reduction is here associated with an enlarged and multilobed lens (arrowhead). C: Extension of retinal pigmented epithelium (RPE) toward the midline is shown by an arrow. D,E: Colobomas is associated with reduction of the anteriorbrain structures (D), which is even more pronounced in E. F: Dorsal view of a Xenopus tadpole injected with xPitx3-EnR in one blastomere at the two-cell stage. Interference with normal xPitx3 activity resulted in the absence of an eye on the injected side.
Figure 7. Whole-mount in situ hybridization shows the effects of overexpression of xPitx3 and xPitx3/engrailed repressor chimeric RNAs (Eng-Pitx3), as well as Pitx3 morpholino oligonucleotides (Pitx3-Mo) on the expression of eye marker genes. The uninjected side of embryos (left side) was used as a contralateral control to compare the gene expression changes in the injected side. A,D,G,J,M: Anterior view of embryos microinjected with xPitx3 wild-type RNA analyzed at the early (A,D,G,J,M) and later (A prime ,D prime ,G prime ,J prime ,M prime ) stages of development. B,E,H,K,N: The expression patterns (anterior view) of these genes at both the early (B,E,H,K,N) and late stages (B prime ,E prime ,H prime ,K prime ,N prime ) were examined upon overexpression of Pitx3/engrailed repressor chimera. C,F,I,L,O: Microinjection of Pitx3-Mo revealed similar effects on eye marker genes in their early (C,F,I,L,O) and later (C prime ,F prime ,I prime ,L prime ,O prime ) expression as demonstrated by Eng-Pitx3 overexpression. Q: Lateral view of tail bud stage embryo showing the effect of the xPitx3-EnR overexpression on Xenopus beta - beta 1-Crystallin expression domain. P: The uninjected control side of the same embryo (lateral view). An increase and reduction of the expression domains of examined genes are shown with black and white arrowheads, respectively.
