The role of Pax-6 in lens regeneration

Abstract

Pax-6 is a master regulator of eye development and is expressed in the dorsal and ventral iris during newt lens regeneration. We show that expression of Pax-6 during newt lens regeneration coincides with cell proliferation. By knocking down expression of Pax-6 via treatment with morpholinos, we found that proliferation of iris pigment epithelial cells was dramatically reduced both in vitro and in vivo, and, as a result, lens regeneration was significantly retarded. However, induction of dedifferentiation in the dorsal iris was not inhibited. Pax-6 knockdown early in lens regeneration resulted in inhibition of crystallin expression and retardation of lens fiber induction. Once crystallin expression and differentiation of lens fibers has ensued, however, loss of function of Pax-6 did not affect crystallin expression and lens fiber maintenance, even though the effects on proliferation persisted. These results conclusively show that Pax-6 is associated with distinct early events during lens regeneration, namely control of cell proliferation and subsequent lens fiber differentiation.

Fig. 1.

Pax-6 is associated with proliferating cells in the dorsal and ventral iris during lens regeneration. Pax-6-expressing cells are shown in blue, and BrdU-positive cells are shown in green. (A, D, and G) BrdU-positive cells are present in both the dorsal iris (di) and the ventral iris (vi) at 5 (A), 15 (D), and 25 (G) days after lentectomy. (B, E, and H) All BrdU-positive cells in the dorsal and ventral iris also express Pax-6 (arrows). (C, F, and I) The percentage of proliferating cells is higher in the dorsal iris at 5 days after lentectomy (C), and this difference increases at 15 (F) and 25 (I) days after lentectomy. (C and F) Proliferation in the dorsal and ventral iris at 5 and 15 days after lentectomy is mostly limited to the central part of the iris and is reduced in the peripheral regions. (G–I) By day 25 after lentectomy the wave of proliferation (G) and Pax-6 expression (H) moves laterally along the lens epithelium (le) of the regenerating lens. lv, lens vesicle.

Fig. 2.

Pax-6 morpholinos decrease levels of Pax-6 and proliferation in cultured PECs. Morpholino fluorescence is red, and Pax-6 immunofluorescence is blue. Untreated cells (A and F) and cells treated with control (C-Mo) (B) or Mis-Mo (C) show robust Pax-6 immunofluorescence, whereas cells treated with morpholinos against Pax-6 (Pax6-Mo1 and Pax6-Mo2) show reduced levels of Pax-6 (D and E). (G–J) Pax-6 immunofluorescence only from B–E. Cultured PECs transfected with Pax-6 morpholinos (M and N) show fewer BrdU-positive cells than cells transfected with the control (K) or the Mis-Mo (L). The number of proliferating cells is shown as a percentage of the number of cells transfected with the morpholinos (O). The reduction in proliferation is significant (P < 0.01). Asterisks in O denote statistical significance. All error bars are SEM.

Fig. 3.

Knockdown of Pax-6 decreases proliferation and inhibits lens regeneration. (A–E) Animals were treated with morpholinos at 10 days after lentectomy and collected at day 13 after lentectomy. Untreated control eyes (A) and eyes treated with C-Mo (B) express similar levels of Pax-6 (blue) in the regenerating lens vesicle. (C) Treatment with Pax6-Mo1 reduces expression of Pax-6. (D and E) Pax-6 staining only from B and C, respectively. (F–I) Animals were injected with morpholinos at day 4 and day 10 after lentectomy and collected at 15 days after lentectomy. (F) Animals treated with Pax-6 morpholinos showed a marked reduction in lens vesicle formation compared with untreated animals or animals injected with the C-Mo or Mis-Mo. (G and H) The lens vesicle from untreated animals and animals treated with C-Mo have elongating cells (Insets). The lens vesicle fails to form in animals treated with Pax6-Mo1; however, dedifferentiation at the dorsal tip does occur (I). (J–N) Animals were injected with morpholinos at 10 day after lentectomy and collected at 13 days after lentectomy. Lens vesicles of untreated control eyes (J) and of eyes treated with C-Mo (K) have a higher number of BrdU-positive cells compared with eyes treated with Pax6-Mo1 (L). (M and N) BrdU staining of K and L, respectively. (O) Morpholino knockdown of Pax-6 results in a statistically significant reduction in proliferation. The number of proliferating cells is shown as a percentage of the total number of cells in each group. All error bars are SEM. Asterisks indicate statistical significance of P < 0.01 when compared with animals treated with C-Mo or Mis-Mo. Sections shown in B and K and in C and L are identical and were stained for Pax-6 and BrdU, but the results are presented separately.

Fig. 4.

Pax-6 morpholino treatment inhibits crystallin expression. Morpholinos were injected at day 10 after lentectomy, and animals were collected for assay at day 13 after lentectomy. (A and F) Lens vesicles from untreated control eyes express α and β crystallin (α-cry or β-cry). (B and G) Eyes treated with C-Mo also express α and β crystallin. (D and I) Lens vesicles from eyes treated with Pax6-Mo1 do not show any α or β crystallin expression. (C, E, H, and J) α or β crystallin staining only from B and D (C and E) and from G and I (H and J).

Fig. 5.

Pax-6 morpholino treatments affect proliferation of lens epithelial cells at later stages of regeneration and have no effects on the regulation of crystallin expression. Morpholinos were injected at day 20 after lentectomy and collected at day 23 after lentectomy. (A and B) Untreated eyes (A) and eyes treated with C-Mo (B) have an equivalent number of proliferating cells. (C) Eyes treated with Pax6-Mo1 show reduced proliferation. (D) There are ≈25% more cells proliferating in the lens epithelium of C-Mo-treated eyes than Pax-6 morpholino-treated eyes. This difference is statistically significant (P < 0.01). (E–G and L–N) α crystallin expression. Eyes treated with C-Mo express α crystallin protein (E and F) and transcripts (G), and these expression patterns are not altered in eyes treated with Pax6-Mo1 (L–N). In G the ripped lens epithelium is a histological artifact. Likewise, there is no alteration in expression patterns for β crystallin protein and transcripts in eyes treated with C-Mo (I–K) or Pax6-Mo1 (P–R). Note that β crystallin is not expressed in the lens epithelium (le). (H and O) Adjacent sections to the sections used for in situ hybridization showing expression of Pax-6 in C-Mo-treated (H) or Pax6-Mo1-treated (O) eyes. Error bars in D are SEM. α-cry, α crystallin; β-cry, β crystallin; lf, lens fibers; le, lens epithelium.