Characterizing the lens regeneration process in Pleurodeles waltl

Abstract

Background: Aging and regeneration are heavily linked processes. While it is generally accepted that regenerative capacity declines with age, some vertebrates, such as newts, can bypass the deleterious effects of aging and successfully regenerate a lens throughout their lifetime.

Results: Here, we used Spectral-Domain Optical Coherence Tomography (SD-OCT) to monitor the lens regeneration process of larvae, juvenile, and adult newts. While all three life stages were able to regenerate a lens through transdifferentiation of the dorsal iris pigment epithelial cells (iPECs), an age-related change in the kinetics of the regeneration process was observed. Consistent with these findings, iPECs from older animals exhibited a delay in cell cycle re-entry. Furthermore, it was observed that clearance of the extracellular matrix (ECM) was delayed in older organisms.

Conclusions: Collectively, our results suggest that although lens regeneration capacity does not decline throughout the lifespan of newts, the intrinsic and extrinsic cellular changes associated with aging alter the kinetics of this process. By understanding how these changes affect lens regeneration in newts, we can gain important insights for restoring the age-related regeneration decline observed in most vertebrates.

Keywords: Aging; Cell cycle; ECM remodeling; Lens regeneration; SD-OCT; iPECs.

Fig. 1:

In vivo monitoring of lens regeneration from larval, juvenile, and adult newts. (A-C) Pictures of larvae, juvenile and adult P. waltl. (D-X) B-scan images are shown here in grayscale images. Eyes were kept at the same orientation for all time points, with cornea (C) at the anterior, dorsal iris (D) on the left side, and ventral iris (V) to the right of the image. This figure depicts the timing of morphological events that occur during lens regeneration in each age group such as healing of cornea wound (CW), extracellular matrix (ECM) remodeling in the anterior (AC) and posterior chambers (PC), lens vesicle (LV) appearance, primary lens fiber (1°LF) differentiation from the epithelial cells of the lens vesicle, and lens epithelial differentiation into secondary lens fibers (2°LF). Approximate lens regeneration stages according to Sato (1940) are depicted on the top right of each OCT image.

Fig. 2:

Three-dimensional view of lens regeneration. B- scans were used to reconstruct a three-dimensional image of the newt eye. To aid in visualization, the regenerating lens was pseudo-colored with red color. Three-dimensional images permit detailed observations of lens growth and morphogenesis. When it first appears, the lens vesicle is located in the mid-dorsal region of the iris and has an asymmetrical shape (A, E, I). As the lens develops and more lens epithelial cells differentiate into lens fibers, the regenerating lens assumes a spherical shape (D, G, H).

Fig. 3:

Comparisons of lens growth rates between three age groups. A: SD-OCT images were used to calculate the volume of lens vesicles from the time they appeared until 48 dpl. The raw volume data is plotted across the day of the experiment. B: A Generalized Additive Mixed Model (GAMM) was used to estimate the effect of the three groups on lens log(Volume) once the lens vesicle appears.

Fig. 4:

Histological examination and quantification of cell cycle dynamics during lens regeneration. A: EdU staining was performed to explore differences in cell cycle re-entrance and progression of the iris pigment epithelium between the three age groups. EdU+ cells were observed in the iris epithelium (dotted line) and stroma of larval eyes at 1 dpl and in higher numbers at 4 dpl. By 10 dpl EdU+ cells were observed in the newly formed lens vesicle. EdU+ cells were found at 4 dpl in juvenile eyes and not until 10 dpl in adult eyes in the iris pigment epithelium. B: The ratio of the number of iris epithelial EdU+ cells to Hoechst+ cells was plotted along the y-axis vs the day of lentectomy on the x-axis. Raw data (points) estimated mean ratio (triangle), and error bars using the standard error of the estimates are all displayed in the graph. Among the eight within-day comparisons with adult eyes, those with small FDR-adjusted p-values are annotated; none of the other three had p-values less than 0.2. For A only the middle cross-section of each eye is shown, whereas in B a total of 9 cross-sections covering the entire area of the eye were used for quantification.

Fig. 5:

Histological examination of ECM remodeling during the early stages of lens regeneration. Picrosirius red staining was performed to visualize collagen composition and distribution throughout the newt eye at different stages of lens regeneration. Breakdown of collagen fibers in the anterior chamber is evident as early as 1 dpl in larval animals and by 10 dpl almost all collagen is cleared out of the eye chamber. On the contrary, collagen staining was observed all over the eye cavity in juvenile and adult eyes at 1 dpl. The remodeling process occurs slower in adult animals, as evidenced by the collagen staining detected in the anterior chamber at 15 dpl. D= dorsal iris; V= ventral iris. Magnifications are shown in each figure (4x: 100 um; 20x: 500 um).

Fig. 6.

Graphical description of the proposed stages of lens regeneration in Pleurodeles waltl. Lens regeneration staging was modified from Sato’s stages (Sato, 1940; Yamada, 1977). Prior to injury iPECs are terminally differentiated adult somatic cells and at rest in G₀. Following lentectomy, the aqueous and vitreous chambers are disrupted by the lentectomy and fill with extracellular matrix (appears as condensed and compact fibers and sheets)[Sato’s stage 0]. Also at this time, immune and blood cells migrate into the injured area. Subsequently, the iPECs re-enter the cell cycle (transition from G₀ to G₁) [Sato’s stage I]. Extracellular remodeling also starts at this time. Soon after, the iPECs at the tip of the dorsal iris become depigmented while extracellular matrix remodeling continues to takes place [Sato’s II]. The depigmented iPECs proliferate and differentiate into lens cells giving rise to an early lens vesicle. These cells start synthesizing lens specific proteins such as Crystallins [Sato’s stage III-IV]. At this time extracellular matrix is mostly removed from the eye chambers. Cells of the lens vesicle continue to proliferate. Cells located in the inner wall of the lens vesicle start to elongate [Sato’s stage IV-V]. The remaining iPECs that were partially depigmented start to withdraw from the cell cycle. As lens morphogenesis continues, internal cells differentiate into primary lens fibers, and the lens epithelial cells on the anterior side proliferate [Sato’s stage VI-VII]. Next, the primary lens fibers start to lose their nucleus and other organelles and assume a concentric arrangement [Sato’s stage VIII]. Lens epithelial cells at the equatorial zone differentiate giving rise to secondary lens fibers. During this time, iPECs cease proliferation and start to resynthesize their melanosomes [Sato’s stage VIII-IX]. Almost all lens fibers lose their nucleus and other organelles, except the newly formed fibers at the outer region near the lens equator. The regenerating lens detaches from the dorsal iris and assumes a position at the center of the eye [Sato’s stage X-XI].