Temporal regulation of planarian eye regeneration

Abstract

While tissue regeneration is typically studied using standard injury models, in nature injuries vary greatly in the amount and location of tissues lost. Planarians have the unique ability to regenerate from many different injuries (including from tiny fragments with no brain), allowing us to study the effects of different injuries on regeneration timelines. We followed the timing of regeneration for one organ, the eye, after multiple injury types that involved tissue loss (single- and double-eye ablation, and decapitation) in Schmidtea mediterranea. Our data reveal that the timing of regeneration remained constant despite changing injury parameters. Optic tissue regrowth, nerve re-innervation, and functional recovery were similar between injury types (even when the animal was simultaneously regrowing its brain). Changes in metabolic rate (i.e., starving vs. fed regenerates) also had no effect on regeneration timelines. In addition, our data suggest there may exist a role for optic nerve degeneration following eye ablation. Our results suggest that the temporal regulation of planarian eye regeneration is tightly controlled and resistant to variations in injury type.

Keywords: Eyes; innervation; nerve degeneration; planaria; regeneration timing.

Figure 1

Planarian model system and microsurgical assays. (A) Planarian anatomy. Dorsal, lateral, and transverse cross‐section views. Cross‐section stained with hematoxylin and eosin (H&E). (B) Diagram of the planarian eye. Optic cup view showing the two eye tissue types, and visual nervous system view showing the optic chiasm (asterisk) and innervation to the brain. (C) Schematic view of surgery set‐up (performed under a dissecting scope). (D) Decapitation assay. Dotted line, amputation plane. (E) Ablation assay. Dotted circle, ablated region.

Figure 2

Timing of eye regeneration does not change with injury type. (A)−(C) Regeneration morphology. The same worms pictured before surgery (intact) and over 28 days of regeneration. Phenotypes shown are representative of n ≥ 40 in each condition. (A) Decapitated, (B) one eye ablated, and (C) both eyes ablated. Note that although eyes regenerate by 14 days in all conditions, they are smaller than intact eyes. Dotted lines, amputation planes; dotted circles, ablated regions. Scale bars: 100 μm. (D) Graph of eye sizes for the entire optic cup and the pigment spot alone compared to the original size prior to eye loss (n ≥ 9). **P < 0.001; *P < 0.01. (E) Cross‐sections (H&Es). Pigment cells, black tissues; Asterisks, brain. Scale bars: 25 μm. (F) Wound size (at 1 h post ablation) relative to both the eye's original size prior to ablation and the regenerated eye at day 14 (n ≥ 10). Insets: wound and regenerated eye in the same animal, with borders outlined in red for clarity. *P < 0.01. Scale bars: 5 μm. (G) Phospho‐histone‐H3 staining at 4 h post ablation (hpa). (G′) Brightfield images of fixed worms in (G), showing eye location/absence. Asterisks, eyes. ^* P < 0.05. Scale bars: 100 μm (n ≥ 4). Red arrows, ablated eyes; yellow arrows, regenerated eyes; dpa, days post amputation/ablation; error bars, standard error of the mean.

Figure 3

Decapitation, but not eye ablation, disturbs underlying brain structures. Anti‐synapsin immunolabeling of the nervous system. (A) Decapitated and (B) one‐eye ablated worms. (B′) Brightfield images of fixed worms in (B), shown to mark ablation site. Brain is removed following decapitation and restored by day 14, whereas the brain is undisturbed following ablation. Brackets, bi‐lobed cephalic ganglia (brain); dotted lines, amputation planes; dotted circle, ablated region; red arrows, ablated eyes; yellow arrow, regenerated eye; dpa, days post amputation/ablation. n = 10. Scale bars: 100 μm.

Figure 4

Timing of eye regeneration does not change with food intake. (A) Starving worms, n ≥ 10. (B) Feeding worms, n ≥ 9. For both conditions, one eye was ablated and regeneration in the same worm was followed for 28 days. Both overall optic cup size (top graphs) and pigment spot size (bottom graphs) were measured, as a percentage of the original eye size (size of the eye prior to its ablation). Control, eyes in non‐ablated worms; intact, non‐ablated eye from ablated worms; ablated, ablated eye from ablated worms; dashed line, control level. (C) Comparison of ablated eyes only. Graphs of just the ablated eye data from (A) and (B). Final 28‐day average optic cup sizes in starved (70.39%) and fed (86.95%) worms were not statistically significant (P = 0.4), nor were average pigment spot sizes (starved 61.92% and fed 81.83%, P = 0.36). Dpa, days post ablation. ^* P ≤ 0.01; ^* P = 0.02. Error bars, standard error of the mean.

Figure 5

Timing of functional eye recovery does not change with injury type. (A) Behavioral phototactic assay. Photophobic planarians avoid traveling through green wavelengths. Examples of (A1) a positive behavioral response, where worms turn away from the light, and (A2) no response, where worms travel directly through the light. (B) Injury types. (B1) Sham ablated, (B2) both eyes ablated, and (B3) decapitated worms at 24 h post injury. Red arrowheads, ablation sites; dotted line, amputation plane; green arrows, pharynx. (C) Behavioral responses. (C1) Positive (green light) and negative (no light) controls using uninjured wildtype worms. Without any light cues, 25% of worms randomly change direction (“response”). (C2) Responses over time. On day 4 of regeneration, both decapitated and double‐eye ablated worms fail to respond to green light (not significantly different from no light controls, P ≥ 0.32). Photophobia reappears starting on day 5, with all worms functionally recovered by day 7. Dpa, days post amputation/ablation. ^* P < 0.01. Error bars, standard error of proportion. n = 20.

Figure 6

Timing of optic nerve regeneration does not change with injury type. (A)−(C) Anti‐arrestin immunolabeling. (A) Decapitated and (B) one‐eye ablated worms both initiate photoreceptor regeneration by day 4, and complete regeneration by day 14. Dotted lines, amputation planes. n = 10. Scale bars: 100 μm. (C)−(E) Optic nerve re‐innervation following ablation. (C) Confocal z‐stacks. At 1 h post ablation the original innervation (C1, white arrow) from the ablated optic cup is still visible, but is gone by day 1 (C2, red arrow). Photoreceptors in the new optic cup reappear by day 2 (C3). Processes extend from both optic cup and chiasm by day 3 (C4), finally meeting on day 4 (C5, yellow arrow). Scale bars: 15 μm. (D) Optic nerve regeneration phenotypes by percentage. Majority phenotype in blue highlight. (E) Diagram of the neural “bulge” (in red). The neural bulge is the last part of the optic nerve to be lost. Red arrowheads, ablated eyes; yellow arrowheads, regenerating eyes.

Figure 7

The timing of pigment cell regeneration parallels the timing of optic nerve regeneration. (A)−(D) Tyrosinase labeling of pigment cells over time. Whole mount in situ hybridizations in one‐eye ablated worms (left eye) through day 4. Normal tyrosinase expression is revealed by the intact (right) eye. Note that pigment cell regeneration does not start until day 2, when photoreceptor cells also begin regenerating. n ≥ 5. Phenotypic penetrance is 100%, except on day 2 (B1, B2). Dpa, days post ablation; red arrows, ablated eye; yellow arrows, regenerating eye. Scale bars: 100 μm.

Figure 8

Model of eye regeneration timeline, with surgical ablation as example injury. Green, visual nervous system; red, ipsi/epsilateral neural bulge; black crescent, pigment cells.