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Few molecular comparisons have been made between the processes of embryogenesis and regeneration or transdifferentiation that lead to the formation of the same structures. In the amphibian, Xenopus laevis, the cornea can undergo transdifferentiation to form a lens when the original lens is removed during tadpole larval stages. Unlike the process of embryonic lens induction, cornea-lens transdifferentiation is elicited via a single inductive interaction involving factors produced by the neural retina. In this study, we compared the expression of a number of genes known to be activated during various phases of embryonic lens formation, during the process of cornea-lens transdifferentiation. mRNA expression was monitored via in situ hybridization using digoxigenin-labeled riboprobes of pax-6, Xotx2, xSOX3, XProx1, and gamma6-cry. We found that all of the genes studied are expressed during both embryogenesis and cornea-lens transdifferentiation, though in some cases their relative temporal sequences are not maintained. The reiterated expression of these genes suggests that a large suite of genes activated during embryonic lens formation are also involved in cornea-lens transdifferentiation. Ultimately functional tests will be required to determine whether they actually play similar roles in these processes. It is significant that the single inductive event responsible for initiating cornea-lens transdifferentiation triggers the expression of genes activated during both the early and late phases of embryonic lens induction. These findings have significant implications in terms of our current understanding of the "multistep" process of lens induction. Dev Dyn 1999;215:308-318.
Fig. 1. Summary of lens induction and cornea-lens transdifferentia- tion. (A) During the early phase of lens induction (gastrula and neural plate stages 11.5–14), inductive signals (dashed arrows) originating from the neural plate act in a planar fashion, and from the underlying mesoderm and endoderm, in a vertical manner, on the presumptive lensectoderm. (B) During the late phase of lens induction the optic vesicle contacts the headectoderm and further signals from the optic cup act on the lensectoderm to pinpoint the site of lens formation (beginning at stage 19). (C and D) The developing lens grows into the cavity that forms in the optic vesicle. (E) The lens eventually detaches from the overlying headectoderm and continues to undergo differentiation in the optic cup. The inner cornea is derived from migrating neural crest cells while the outer cornea is derived from the remaining headectoderm overlying the eyecup. (F) The cornea of a premetamorphosed tadpole has the ability to undergo transdifferentiation to form a new lens if the original lens is removed during larval stages. (G) Signals from the neural retina (dashed arrow) act on the inner layer of the outer cornea where the new lens initially forms (H). (I) This tissue ultimately develops into a fully differenti- ated lens, which resides in the optic cup. All stages follow those of Nieuwkoop and Faber (1956).
Fig. 2. In situ expression patterns of Xenopus pax-6 (A–G) and Xotx2 (H–K) mRNA. (A) Leftlateral view of the head of a stage-32 embryo. pax-6 expression is seen in the fore- and mid-brain, retina and lensepithelium (expression appears to be absent from the central lens fiber cells). (B–G) Lateral views of lentectomized and unoperated larval eyes. (B) Expression of pax-6 can be seen in about half of the 4-day transdifferentiating cornea overlying the pupillary space. (C) After 5 days expression expands and fills the entire area over the pupillary space. (D) By 8 days corneal expression becomes focused to a tight spot, which in this case is located at the edge of the pupillary space, indicated by the white arrow head. Even though we examined albino frogs, dark brown pigment is normally present in the eye (pigmented epithelium). Condensa- tions of natural brown pigment lying just outside the collapsed pupillary space are indicated by black arrowheads. This pigmentation can be readily distinguished from hybridized probe. (E) A typical unoperated righteye exhibits no hybridization of the anti-sense RNA probe. (F) Example of a 5-day transdifferentiating cornea hybridized with sense-control probe also exhibits no hybridization. (G) Example of a younger, 2 day transdiffer- entiating cornea hybridized with anti-sense probe, which exhibits no expression of pax-6. (H) Leftlateral view of the head of a stage-32 embryo. Expression of Xotx-2 is seen in the lens placode, optic vesicle and the developing brain. (I–K) Leftlateral views of lentectomized larval eyes. (I) Example of an eye exhibiting positive expression in a 6-day transdifferentiating cornea. Expression appears as a solitary spot overly- ing the pupillary space, indicated by the white arrowhead. (J) Example of a 6-day transdifferentiating cornea exhibiting no hybridization of the sense-control probe. (K) Example of a younger, 2-day transdifferentiating cornea hybridized with anti-sense probe, which exhibits no expression of xOtx2. Partial dotted lines in B–G, and I–K are included to help demarcate the perimeter of the pupillary space. Abbreviations: rt, retina; le, lens; ps, pupillary space; lp, lens placode. Scale bar: 200 μm (the scale bars in G and K apply to A–G; and H–K, respectively).
Fig. 3. In situ expression patterns of xSOX3 (A–D), XProx1 (E–H), and gamma-cry (I–M) mRNA. (A) Leftlateral view of the head of a stage-32 embryo. Strong xSOX3 expression is observed throughout the cns and lens. Other areas of expression include what are presumed to be derivatives of the unidentified dorsolateral cranial placodes (black arrow- head). (B–D) Leftlateral views of lentectomized larval eyes. (B) Example displaying xSOX3 expression in a 2-day transdifferentiating cornea is seen as a solitary spot centrally located over the pupillary space, indicated by a white arrowhead. (C) Example of a 5-day transdifferentiating cornea that exhibits only background levels of hybridization of the sense-control probe. (D) Example of a younger, 2-day transdifferentiating cornea hybridized with anti-sense probe, which exhibits no expression of xSOX3. (E) Leftlateral views of, from left to right, stage 28, 32, and 35 embryos, with the anterior end facing upwards. Expression is first observed broadly within the lens placode, which later becomes restricted to the developing lens. (F–H) Leftlateral views of lentectomized larval eyes. (F) Case exhibiting corneal expression in a solitary spot centrally located over the pupillary space after 8 days of transdifferentiation, indicated by a black arrowhead. Naturally occurring dark brown pigment within the pigmented epithelium condenses at the edges of the collapsed pupillary space (white arrowheads). (G) Example of a 6-day transdifferentiating cornea hybrid- ized with sense-control probe demonstrating no detectable hybridization in the cornea. (H) Example of a younger, 2-day transdifferentiating cornea hybridized with anti-sense probe, which exhibits no expression of XProx1. (I) Leftlateral view of the head of a stage-30 embryo. Strong expression of ô°€6-cry can clearly be seen in the developing lens. (J–L) Leftlateral views of lentectomized larval eyes. (J) Example displaying corneal expression centrally located over the pupillary space in a 7-day transdifferentiating cornea, as indicated by the black arrowhead. (K) Example of a 6-day transdifferentiating cornea exhibiting no hybridization with sense-control probe. (L) Example of a younger, 1-day transdifferentiating cornea hybridized with anti-sense probe, which exhibits no detectable expression of gamma-cry. Partial dotted lines in B–D, F–H, and I–L are included to help demarcate the perimeter of the pupillary space. Abbreviations: cns, central nervous system; lt, lentoid; other abbreviations are as indicated in Figure 2. Scale bar: 200 μm (A, C–D, F–L); 330 μm (B); 800 μm (E).
cryga (crystallin gamma A) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anteriorleft, dorsal up.
