Latent regeneration abilities persist following recent evolutionary loss in asexual annelids

Abstract

Regeneration abilities have been repeatedly lost in many animal phyla. However, because regeneration research has focused almost exclusively on highly regenerative taxa or on comparisons between regenerating and nonregenerating taxa that are deeply diverged, virtually nothing is known about how regeneration loss occurs. Here, we show that, following a recent evolutionary loss of regeneration, regenerative abilities can remain latent and still be elicited. Using comparative regeneration experiments and a molecular phylogeny, we show that ancestral head regeneration abilities have been lost three times among naidine annelids, a group of small aquatic worms that typically reproduce asexually by fission. In all three lineages incapable of head regeneration, worms consistently seal the wound but fail to progress to the first stage of tissue replacement. However, despite this coarse-level convergence in regeneration loss, further investigation of two of these lineages reveals marked differences in how much of the regeneration machinery has been abolished. Most notably, in a species representing one of these two lineages, but not in a representative of the other, amputation within a narrow proliferative region that forms during fission can still elicit regeneration of an essentially normal head. Thus, the presence at the wound site of elements characteristic of actively growing tissues, such as activated stem cells or growth factors, may permit blocks to regeneration to be circumvented, allowing latent regeneration abilities to be manifested.

Fig. 1.

Fission and comparative regeneration experiments in naidine annelids. In this and subsequent figures, anterior is left; dark-green and light green mark new head and tail tissue, respectively, formed by fission; dark-gray bars mark the original body region remaining after amputation; light-gray bars mark amputated tissue; orange bars mark regenerated tissue; dashed lines indicate plane of amputation. (A) Naidine paratomic fission occurs by intercalation of new head and tail tissue in the middle of the body (top: prefission; middle: fission; bottom: post-fission). (B) Pa. litoralis individual with a late-stage fission zone, showing the characteristically clear tissue of this zone. Original head is at top, pointing left. (Scale bar, 250 μm.) (C–F) Pr. leidyi can regenerate anteriorly and posteriorly (C and D: 4 days dpa), whereas Pa. litoralis can regenerate posteriorly but not anteriorly (E and F: 7 dpa). (G) Results from comparative regeneration experiments (19 naidine species, top; 2 outgroups, bottom). See Table S2 for amputation locations for each species. Color codes are based on the stage of the most advanced three individuals, scored at the end of that time period, with emergence of chaetae from new segments marking regeneration completion. Absence of regeneration scoring indicates that no individuals survived to that time point.

Fig. 2.

Phylogenetic distribution of head regeneration ability in naidines. Open circles mark species that can regenerate a head; solid circles mark species that cannot. Head regeneration appears to have been lost three times within one of the two naidine clades that undergo fission (green ovals). Regeneration information is based on our comparative regeneration experiments and previously published data for Ripistes parasita (21). Tree topology is from a Bayesian analysis. Nodes with a posterior probability of 0.98 or greater are black; those with a posterior probability less than 0.98 are gray. All posterior probabilities are 1.0 unless denoted by a letter (a: 0.99; b: 0.98; c: 0.95; d: 0.89; e: 0.86; f: 0.82; g: 0.56). Maximum likelihood tree has an identical topology and nearly identical branch lengths, although weak bootstrap support.

Fig. 3.

Minimal amputation experiments and post-amputation cell proliferation. Body region indicators are as in Fig. 1; arrows mark the prostomium; asterisks mark the mouth. (A–C) Head morphology of uncut worms. (D–I) Following amputation of the asegmental tip, Pa. litoralis (D, 4 dpa) and Pr. leidyi (F, 3 dpa) can regenerate this region but C. diaphanus merely wound heals (E, 7 dpa, showing the characteristic square anterior margin following within-head amputation in this species). Following amputation of one head segment and the asegmental tip, Pa. litoralis (G, 7 dpa) and C. diaphanus (H, 7dpa) only wound heal, but Pr. leidyi regenerates this region (I, 3 dpa; caret marks ventral chaetae of regenerated segment). (J–O) BrdU labeling on days 2 and 3 following head amputation (removing three segments in Pa. litoralis, four in C. diaphanus, and six in Pr. leidyi) indicates proliferation at the wound site in Pa. litoralis (J and M: solid arrowheads) even though no blastema will form; no proliferation at all at the C. diaphanus wound site (K and N: open arrowheads); and extensive proliferation associated with the developing blastema of Pr. leidyi (L and O: solid arrowheads). Right panels in K and N are the BrdU-labeled tails (undergoing normal growth) of the anteriorly amputated individuals pictured in the associated left panels.

Fig. 4.

FZ-regeneration following fission-zone head bisection. Body region indicators are as in Fig. 1; dark-green and light-green bars mark new head and tail tissue, respectively, formed in the fission zone; paired vertical dashes mark the boundary between original segments and fission zone tissue; asterisks mark chaetae of remaining fission zone segment (segment 3 in Pa. litoralis). Labeling of structures: mouth (m), prostomium (pr), cerebral ganglion (cg), pharynx (ph), segment 1 chaetae (ch1), ventral ganglion of segment 1 (g1). (A–C) Fission-zone head bisection, diagrammed in A (A', Pa. litoralis late-stage fission zone), leads to replacement of head structures in Pa. litoralis (B, 8 dpa) but only wound healing in C. diaphanus (C, 6 dpa). Insets in B show the chaetae of segment 1 (left) and of the remaining fission zone segment (right) from more superficial focal planes. (D–F) FZ-regeneration in Pa. litoralis occurs by the formation of a blastema that shows extensive cell proliferation and actively grows. BrdU labeling (D, 4 dpa) is widespread throughout the blastema epidermis (inset in D is high-magnification view of chaeta from fission zone segment). Live imaging of the same FZ-regenerating Pa. litoralis individual at 7 dpa (E) and 15 dpa (F) shows blastema growth. (G and H) FZ-regeneration in Pa. litoralis (H, 10 dpa) restores internal and external structures present in a normal head (G). Specimens are labeled for acetylated α-tubulin (green, labeling peripheral nerves and cilia of the foregut, prostomium, and body wall), for serotonin (red, labeling serotonin⁺ nerve cells and tracts), and with phalloidin (blue, labeling the body-wall muscles to show the body contour). Note that the ventral mouth and buccal cavity are unciliated and are thus unlabeled. Segmental chaetae are autofluorescent. Inset in H shows segment 1 chaetae. Abnormalities in the FZ-regenerate in H include a misshapen pharynx, supernumerary serotonin⁺ ganglion cells, a laterally rotated cerebral ganglion, and absence of segment 2 (or at least its chaetae). (I) Pr. leidyi nanos is expressed throughout the anterior blastema during regular regeneration (2 dpa). (J–L) Pa. litoralis nanos is not expressed during failed regeneration after removal of two adult head segments (J, caret marks segment 3 chaetae), but is expressed in the FZ-regeneration blastema following amputation of these same segments from within the fission zone (K, 4 dpa; L, 5 dpa).