The history and enduring contributions of planarians to the study of animal regeneration

Abstract

Having an almost unlimited capacity to regenerate tissues lost to age and injury, planarians have long fascinated naturalists. In the Western hemisphere alone, their documented history spans more than 200 years. Planarians were described in the early 19th century as being 'immortal under the edge of the knife', and initial investigation of these remarkable animals was significantly influenced by studies of regeneration in other organisms and from the flourishing field of experimental embryology in the late 19th and early 20th centuries. This review strives to place the study of planarian regeneration into a broader historical context by focusing on the significance and evolution of knowledge in this field. It also synthesizes our current molecular understanding of the mechanisms of planarian regeneration uncovered since this animal's relatively recent entrance into the molecular-genetic age.

FIGURE 1. Ancient Japanese texts describe planarians and their ability to fission

1A: Wood-print adapted from the Japanese encyclopedia Kinmô-Zui by Tekisai Nakamura, 1666. Image depicts a striped land planarian (likely Bipalium). The caption indicates that “it is very poisonous and similar to another soil insect (nematode) previously described.” (Translation assistance provided by Dr. Tamaki Suganuma, Nobuo Ueda, and Shigeki Watanabe.) 1B: Wood-print adapted from the illustrated Japanese encyclopedia Wakan Sansai-Zue by Ryōan Terajima, 1713. Image depicts a striped land planarian (likely Bipalium) in the left column. The vertical text is translated to read:

“‘Doko’ or ‘Toku’. The animal has the shape of a Japanese belt in general appearance and is without legs. It measures up to 12 to 15 cm in length; a large specimen attains about 30 cm. The body is flattish in shape as a leaf of leek. There are yellow and black folds on the dorsal surface. The animal has a head shaped like a Japanese forceps…If the animal is touched, fission may occur…”

FIGURE 2. Planarian anatomy is sufficiently complex for regeneration studies

2A: Two depictions of planarian anatomy adapted from Leuckart’s zoological wall chart series entitled “Vermes,” circa 1890. (Image obtained from MBLWHOI Library, Rare Books Archive) 2B: A live planarian extruding its pharynx (arrowhead). Scale bar 200 um. (All animals depicted in Figures 2–7 are the asexual strain of Schmidtea mediterranea unless otherwise noted.) 2C: Overlay of gut (blue, Smed-porcn-1), neurons (yellow, Smed-PC-2), axons, and pharynx (magenta, anti-α-tubulin antibody). Scale bar 200 um. 2D: Left panel: Head of a live planarian. Photoreceptors are darkly pigmented. Right panels: A different specimen showing neurons of the cephalic ganglia (blue, Smed-PC-2), photoreceptors, and commissural visual axons (red, anti-arrestin antibody; a kind gift of Dr. Kiyokazu Agata). Scale bars 200 um. 2E: Tufts of ventral cilia (yellow, anti-acetylated-tubulin antibody) projecting from epithelial cells (nuclei: magenta, TOPRO-3) facilitate swimming. Image focused around opening to the pharynx cavity (M, mouth). Scale bar 50 um. 2F: Left panel: Protonephridia, which compose the excretory system (Smed-innexin-10). Scale bar 200 um. Right panel: Close up of tail tip of a different specimen. Confocal maximum projection of protonephridial system, including flame cells (blue, anti-α-tubulin antibody), proximal tubules (magenta, Smed-innexin-10), and distal tubules (green, Smed-CAVII-1). Scale bar 50 um. (Images provided by Hanh Thi-Kim Vu.) 2G: Markers labeling distinct body regions. Left to right: anterior cells and distal tip of pharynx (Smed-sfrp-1), posterior cells (Smed-wnt11-2), body periphery (Smed-wnt5), and midline (Smed-slit-1).

FIGURE 3. Upon injury, planarians regenerate lost tissues, re-establish scale and proportion, and maintain axial polarity

3A: T.H. Morgan amputated an adult planarian (red dashed line) and observed it regenerate missing anatomy (“epimorphosis”) and re-establish proper body proportions (“morphallaxis”). (Adapted from Morgan, 1900) 3B: A live intact planarian was amputated (white dashed line), and the regenerating tail fragment is shown at 1, 4, and 7 dpa. Scale bar 200 um. dpa: days post amputation. 3C: The cephalic ganglia (arrowheads), pharynx (yellow asterisk), and anterior gut branch (arrow) regenerate by 7 dpa. An intact planarian (left) was amputated (white dashed line) and regenerating tail fragments were stained at timepoints indicated (right) for nervous system, pharynx (green, anti-α-tubulin antibody) and gut (Smed-porcn-1). Scale bars 200 um. (Adapted from Gurley, et al., 2010) 3D: The A/P decision is made by 1 dpa, preceding tissue regeneration and anatomical remodeling. Regenerating tail fragments stained at timepoints indicated for marker of anterior cell identity (Smed-sfrp-1). Scale bars 200 um. hpa= hours post amputation (Adapted from Gurley, et al., 2010)

FIGURE 4. Anterior-posterior polarity

4A: Randolph showed that a small piece of tissue amputated from the flank of the body (left, red box) maintains axial polarity during regeneration (right). (Adapted from Randolph, 1897) 4B: Transplanting tissue from the anterior region of one planarian to a posterior region of another (left) results in outgrowth of a new body axis (right). (Adapted from Santos, 1929) 4C: Thin transverse amputations (left, red dashed lines) cause heteromorphic regeneration, resulting in double-headed (top) or double-tailed (bottom) regenerates. (Adapted from Morgan, 1904) 4D: RNAi strategy employed for Figures 4–6. Animals were 1) fed dsRNA to knockdown a gene of interest, 2) amputated, and 3) allowed to regenerate. 4E: Wnt/β-catenin signaling controls A/P polarity. Live images and fixed animals stained for the nervous system (Smed-PC-2), anterior cell identity (Smed-sfrp-1), and posterior cell identities (Smed-fz-4). Controls regenerate normally. Smed-β-catenin-1(RNAi) causes a head to regenerate from posterior blastemas. Smed-APC-1(RNAi) causes a tail to regenerate from anterior blastemas. Scale bars 200 um. (Live images provided by Dr. Kyle A. Gurley and Dr. Jochen C. Rink.)

FIGURE 5. Dorsal-ventral polarity

5A: Santos showed that flipping the D/V orientation of a tissue plug without altering the A/P orientation results in the outgrowth of an ectopic body axis. This ectopic growth has inverted D/V polarity compared to the main body’s D/V axis. Only the tail region is pictured in Santos’ sketch; the main body’s head is to the left, out of view.(Adapted from Santos, 1931) 5B: BMP signaling controls D/V polarity. Control tail fragments form a blastema (bracket) and regenerate nerve cords localized ventrally only (Smed-PC-2, red arrowheads; compare ventral vs. dorsal views). Smed-smad4(RNAi) causes a loss of blastema formation, regeneration of the cephalic ganglia (compare brackets), and growth of ectopic dorsal nerve cords (black arrowheads; compare ventral vs. dorsal views). Scale bars 200 um. 5C: BMP signaling is required for blastema formation and organization of the midline. Control animals form anterior and posterior blastemas (white arrowheads) and regenerate a midline (Smed-slit-1). Smed-bmp4(RNAi) causes midline indentations in the blastemas, dorsal ruffling (red arrow), and ectopic expression of a midline marker (black arrowheads). Smed-smad4(RNAi) causes a loss of blastema formation (white arrowheads), photoreceptor regeneration in old tissue (white arrows), and ectopic expression of a midline marker (black arrowheads, compare insets). Scale bars 200 um.

FIGURE 6. Medial-lateral polarity

6A: Randolph showed that a midline incision (red dashed line) that is allowed to heal together causes, with some frequency, a duplication of midline structures. (Adapted from Randolph, 1897) 6B: Live images 14 days after the midline incision depicted in 6A. Although the tissue was allowed to heal back together, the head is duplicated at the site of the incision (white arrowhead). The tail is forked (white arrow), even though the posterior was never injured. Species Dugesia sanchezi. Scale bar 200 um. 6C: Smed-slit-1 maintains the M/L axis. Compared to control(RNAi), Smed-slit-1(RNAi) causes the cephalic ganglia, nerve cords (magenta, Smed-PC-2; blue, anti-α-tubulin antibody), photoreceptors (black arrowhead), and markers for the body periphery (Smed-wnt5; compare insets) to collapse towards the midline. Scale bar 200 um. (Adapted from Gurley, et al., 2010) 6D: Smed-wnt5 maintains the M/L axis. Compared to control(RNAi), Smed-wnt5(RNAi) causes the lateral expansion of the axon tracts, and the formation of an ectopic lateral pharynx (blue, anti-α-tubulin antibody; white arrowhead) flanked by gut branches (magenta, Smed-porcn-1). Expression of a midline marker expands laterally (Smed-slit-1; compare insets). Scale bar 200 um. (Adapted from Gurley, et al., 2010)

FIGURE 7. Neoblasts as agents for regeneration

7A: Classical depiction of neoblasts by histology. (Adapted from Stéphan-Dubois, 1965) 7B: Neoblasts (green, Smed-piwi-1) distributed throughout the parenchyma in between gut branches (blue, Smed-porcn-1) and in proximity to the nerve cords (magenta, anti-α-tubulin antibody). Images are confocal maximum projections. Scale bars 50 um. 7C: Irradiation disrupts physiological regeneration. Representative intact planarians at specified dpi, exposed to 10,000 rads from a cesium source. Head regression is observed by 10 dpi, followed by ventral curling around 20 dpi. Lysis generally occurs after 20 dpi. Scale bar 200 um. dpi: days post irradiation 7D: Irradiation eliminates neoblasts and proliferation. Left panels: Neoblasts (green, Smed-piwi-1) are the only mitotic cells (magenta, anti-H3P antibody). White arrowheads indicate examples of colocalization in the tail of a different animal. Right panels: Neoblasts and mitotic cells are eliminated after irradiation by 3dpi. Images are confocal maximum projections. Scale bars 200 um. (Images provided by Dr. Kyle A. Gurley) 7E: Irradiation disrupts restorative regeneration. Top panels: Representative control trunk fragments displaying unpigmented regeneration blastemas by 5 dpa (white arrowheads). Bottom panels: Representative irradiated trunk fragments do not form blastemas (white arrowheads) or regenerate new tissues. Fragments curl ventrally and eventually lyse around 13 dpi. Scale bars 200 um. 7F: Amputation induces two waves of cell proliferation. Mitotic cells (white, anti-H3P antibody) are visualized in regenerating head fragments. A global burst in proliferation is observed within 6 hpa. By 2 dpa, a second proliferative burst occurs at the wound site (yellow arrowheads). Scale bar 200 um.