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Despite extensive study following the pioneering work of Spemann on lens development (Spemann, H. (1901) Verh. Anat. Ges. 15, 61-79) and the subsequent establishment of the concept of embryonic induction, the molecular mechanism of vertebrate lens induction remains largely unknown. Here we report that in Xenopus expression of Pax-6 results in lens formation in a cell autonomous manner. In animal cap experiments, Pax-6 induced expression of the lens-specific marker beta B1-crystallin without inducing the general neural marker NCAM. Ectopic Pax-6 expression also resulted in the formation of ectopic lenses in whole embryos as well as in animal cap explants indicating that in vertebrates, as well as Drosophila (Halder, G., Callaerts, P., and Gehring, W.J. (1995) Science 267, 1788-1792), Pax-6 can direct the development of major components of the eye. Interestingly, ectopic lenses formed in whole embryos without association with neural tissue. Treatments giving rise to anterior neural tissue in animal cap explants resulted in the expression of both beta B1-crystallin and Pax-6. Given the ability of Pax-6 to direct lens formation, we propose that the establishment of Pax-6 expression in the presumptive lensectoderm during normal development is likely to be a critical response of lens-competent ectoderm to early lens inducers.
FIG. 1. Anti-bovine b-crystallin antibody labeling of tadpole stage 38 embryo (A) and close-up (B) of the normal lens from an injected embryo. (C) Transverse section of anti-bovine- b-crystallin anti body-stained tadpole, stage 28. (D)Whole mount in situ hybridization of a stage 28 embryo using a Xenopus bB1-crystallin probe. (E) Animal cap assay of Pax-6-injected Xenopus embryos assessed for the expression of tissue-specific markers at stage 34 using RT�PCR. The lens-specific marker bB1-crystallin was produced in a dose-dependent manner (lanes 1� 4: 1.0, 0.25, 0.06, and 0.5 ng/embryo, respectively) in the absence of NCAM expression. Injection of RNA-encoding Noggin (lane 5, 2.0 ng) serves as a positive control for response of the explants and gives the expected expression of the neural marker NCAM. Muscle actin is used as a mesodermal marker and EF1a as a loading control. (F) Uninjected animal cap stained with b-crystallin antibody. (G, H) Animal caps injected with 1.4 ng Pax-6-FLAG RNA (sibling control at stage 33/34).
FIG. 2. Pax-6 induces ectopic lenses in whole embryos in a cell autonomous fashion. b-Crystallin staining of embryos (stage 38) injected
with RNA-encoding Pax-6-FLAG (0.12� 0.25 ng/embryo) revealed the formation of ectopic lenses (brown) in whole mount (A�C) and in
section (D �G). Red arrowheads (B, F, and G) indicate the border between lensepithelium and fibre cells; large arrowhead in B indicates
endogenous lens. The embryo in A was cleared in benzyl benzoate and is at a higher magnification in B. Embryos shown in C and D were
costained (blue) with antibodies to NCAM. Some ectopic lenses were not associated with neural tissue (C, black arrowhead; D and E)
while others were close to (D, far left ectopic lens; F) or tightly associated with neural tissue (C, white arrowhead; G). Sections through
endogenous (H, left) and ectopic lenses (H, arrowheads, and I) in embryo coinjected with b-Gal and b-crystallin RNAs. Coincident staining
for b-Gal (blue) and b-crystallin (brown) indicates the cell-autonomous function of Pax-6. Magnifications (A) 150, (B, C) 1200, (D) 1100,
and (E� I) 1400.
FIG. 3. Neuralizing treatments result in the expression of Pax-6 and lens-specific bB1-crystallin in animal caps. Injection of RNA-encoding dominant-negative type I BMP receptor (lane 1, 2.0 ng), follistatin (lane 2, 2.0 ng), noggin (lane 3, 2.0 ng), chordin (lane 4, 2.0 ng), dissociated animal cap cells (lane 6), but not uninjected embryos (lane 5) led to the expression of NCAM, Pax-6, and bB1-crystallin in animal caps at tailbud stage. EF-1a was amplified as a loading control and the absence of muscle actin indicated that mesoderm was not present. RNA from tailbudembryo was used as a positive control while an amplification without RT (lane 7) controlled for the absence of contaminating DNA.
crybb1 (crystallin, beta B1 ) gene expression in Xenopus laevis embryos, NF stage 26, as assayed by in situ hybridization. Lateral view: anteriorleft, dorsal up.
crybb1 (crystallin, beta B1 ) gene expression in Xenopus laevis embryos, NF stage 38, as assayed by in immunohistochemistry. Lateral view: anteriorleft, dorsal up.
