FGF signaling is required for lens regeneration in Xenopus laevis

Abstract

In species of the frog genus Xenopus, lens regeneration occurs through a process of transdifferentiation, in which cornea epithelial cells presumably undergo dedifferentiation and subsequently redifferentiate to form a new lens. Experimental studies have shown that the retina provides the key signal required to trigger this process once the original lens is removed. A previous study showed that addition of an exogenous fibroblast growth factor (i.e., FGF1 protein) could initiate transdifferentiation of cornea epithelial cells in culture. To determine the role of FGF signaling in X. laevis lens regeneration, we have examined the presence of specific FGFs and their receptors (FGFRs) during this process and evaluated the necessity of FGFR signaling. Reverse transcriptase-polymerase chain reaction analyses reveal that a number of FGF family members are expressed in cornea epithelium and retinal tissues both before and during the process of lens regeneration. Of these, FGF1, FGF8, and FGF9 are expressed principally in retinal tissue and not in the cornea epithelium. Hence, these ligands could represent key signaling factors originating from the retina that trigger regeneration. The results of experiments using an in vitro eye culture system and an FGFR inhibitor (SU5402) suggest that FGFR signaling is required for lens regeneration in Xenopus.

Figure 1. in vitro eye culture

in vitro eye culture system used to assay lens regeneration in stage 47–49 larvae of X. laevis. (A) The larval eye is shown with both the inner cornea and outer cornea intact. (B) The lens is removed following incision of the outer and inner corneas. (C–D) The outer cornea is tucked into the vitreous chamber of the enucleated eye. (E) The eye is excised from the tadpole and cultured in modified L-15 media with or without FGFR inhibitor (SU5402). Structures are as labeled.

Figure 2. RT-PCR expression of FGFs in eye tissues

Expression of FGFs as determined by RT-PCR assays. Total RNA was collected from corneas and retinas of both non-regenerating control and lens-regenerating larvae. Regenerating corneas and retinas were collected at four timepoints (1, 3, 5, and 7 days after lens removal). Note that the bands for FGF1, FGF8, and FGF9 are uniformly less intense in the cornea when compared to the retina, as determined from replicate RT-PCR reactions. 0d denotes non-regenerating control eye tissues; + denotes positive control cDNA derived from mRNA of whole embryos (st. 37–38); - denotes the negative control without addition of template cDNA.

Figure 3. RT-PCR expression of FGFRs in eye tissues

Expression of FGFRs as determined by RT-PCR assays. Total RNA was collected from corneas and retinas of both non-regenerating control and lens-regenerating larvae. Regenerating corneas and retinas were collected at four timepoints (1, 3, 5, and 7 days after lens removal). 0d denotes non-regenerating control eye tissues; + denotes positive control cDNA derived from mRNA of whole embryos (st. 37–38); − denotes the negative control without addition of template cDNA.

Figure 4. Sectioned eyes after SU5402 treatment in culture

Development of control and SU5402 treated in vitro eye cultures. SU5402 inhibits FGFR function by competitively binding to the FGFR kinase domain. The left column shows sections of representative eyes imaged using differential interference contrast. The right column shows each corresponding fluorescent image illustrating α-lens antibody staining. SU5402 concentrations: (A–B) 0μM control; (C–D) 2μM; (E–F) 5μM; (G–J) 10μM; (K–L) 25μM. The single case of lens regeneration with 10μM SU5402 is shown in G–H. The typical results of non-regenerating cases are shown for 10μM SU5402 (H–I) and 25μM SU5402 (K–L). Arrows point to regenerated lenses; scale bar equals 200μm.

Figure 5. Lens regeneration rates upon application of SU5402

Results of in in vitro eye culture experiments treated with SU5402 to inhibit FGFR function. As shown here, the IC₅₀ for inhibiting lens regeneration is close to 5μM SU5402. Numbers of regenerated lenses and eyes examined are located above each bar; y-axis indicates lens regeneration rate; error bars denote Wilson score intervals in which Z=1.