Vertebrate-like regeneration in the invertebrate chordate amphioxus

Abstract

An important question in biology is why some animals are able to regenerate, whereas others are not. The basal chordate amphioxus is uniquely positioned to address the evolution of regeneration. We report here the high regeneration potential of the European amphioxus Branchiostoma lanceolatum. Adults regenerate both anterior and posterior structures, including neural tube, notochord, fin, and muscle. Development of a classifier based on tail regeneration profiles predicts the assignment of young and old adults to their own class with >94% accuracy. The process involves loss of differentiated characteristics, formation of an msx-expressing blastema, and neurogenesis. Moreover, regeneration is linked to the activation of satellite-like Pax3/7 progenitor cells, the extent of which declines with size and age. Our results provide a framework for understanding the evolution and diversity of regeneration mechanisms in vertebrates.

Fig. 1.

Amphioxus shows extensive regenerative ability. (A) Distribution of regenerative ability in deuterostomes. Regeneration characters are plotted for each phylum and color-coded; regenerative ability of the CNS, organizational level (level), and developmental stage (stage) are indicated. Invertebrates are characterized by extensive (E) regenerative potential compared with the more modest (M) ability seen in vertebrates, with some craniate lineages having particularly limited (L) regeneration capacity. The basal chordate amphioxus regenerates well (asterisk and pink box), in line with other invertebrate deuterostomes. Coding of characters is based on refs. , , and –. (B) Amphioxus juvenile. Amputation levels (dotted lines) and resulting regenerates (≥4 wk, 1–7). The amputation plane is marked by a fine dotted line. Anterior amputation regenerated notochord (white arrowhead) and fin (1 and 2) as well as neural structures and buccal cilia (2, blue arrowhead, and 3). At the level of the velum, a muscular septum separating the buccal and branchial cavities, only wound healing occurred with eventual pharynx degeneration (3, white arrowhead, and 4). Amputation through the pharynx is fatal for both halves (red interval and 5). On sectioning the hepatic diverticulum, a small posterior tail forms (5, blue arrowhead, and Fig. S1), and both the atrium and intestine regenerate. The intestine (6) and a well-proportioned tail (7) regenerate when amputated distally. af, Anterio fin; an, anus; at, atrium; bb, branchial basket; bc, buccal cilia; cf, caudal fin; hd, hepatic diverticulum; in, intestine; mo, mouth; mu, muscle; no, notochord; nt, neural tube.

Fig. 2.

Tail regeneration follows distinct stages. (A) Morphological series in a representative small regenerating adult from day 0 to 15 wk. Polarized light shows regenerated muscle fibers. (B) Sectioning and Mallory triple staining show histological features at stages equivalent to those shown in A. Connective and neural tissues are blue, and striated muscle and notochord are red/purple. (B, far left panel) Cross-section of postanal tail. (B, week 7) Distal reorganization of notochord in week 7 regenerate (black arrow). (B, center panels) Section plane along the base of the neural tube and coronally through the top of the notochord. Vertical white (A) and black (B) dotted lines indicate the amputation plane. Yellow arrows show apparent dedifferentiation zones. (C) Summary of A and B and proposed staging system; a and b are substages of stage 1. All views are lateral, except where stated otherwise. (Scale bar: 100 μm.)

Fig. 3.

Tail regeneration curves differ in large and small amphioxus. Regenerative growth distributions have different shapes for (A) small (red; n = 48) and (B) large (blue; n = 19) amphioxus. The dashed vertical lines indicate breaks in the timeline between days 70 and 105 postamputation (dpa). Growth intervals: small: F(11, 432) = 68.43, P < 0.00001; large: F(11, 155) = 9.70, P < 0.00001. (C) A seven timepoints-based classifier (14–56 dpa) assigns large and small animals to correct size classes with >93% (small, red; n = 48) and >95% accuracy (large, blue; n = 16). Only animals with complete datasets were used for classifier development (n = 64). The graphs display the natural logarithms of the Mahalanobis distances [ln(d₁) vs. ln(d₂)]; the dotted line indicates the optimal class separator.

Fig. 4.

Conservation of molecular markers during tail regeneration. (A) msx is expressed in the mesenchyme of the blastema (stage 2) as well as the overlying wound epithelium. (B) chordin is reexpressed in undifferentiated notochordal cells (stage 3). (C) Confocal image of stage 3 blastema, with acetylated tubulin (AcTub) staining axons of the regenerating neural tube and ciliated cells of the mesenchyme (red arrow). The terminal ampulla is outlined with a dotted line. notochord cells lack cilia. (D) Neurogenesis accompanies axonogenesis, shown by soxB2 expression in the elongating ependymal tube. For clarity, expression patterns are summarized to the right. Black arrow indicates the amputation plane. All sections are represented with anterior to the left and dorsal to the top. no, notochord; nt, neural tube. *Space at intersection of mature and regenerative notochord. (Scale bar: 100 μm.)

Fig. 5.

Expression of Pax3/7 in satellite-like muscle cells during amphioxus regeneration. (A, A′, and A″) Phalloidin labels F-actin in superficial lateral muscle fibers (A) and deeper trails of cells at degrading myosepta (A′) at stage 2 (white arrows). (A″) Overlay with Nomarski optics. (B and B′) More medially, Pax3/7⁺ cells are located in (B) degrading muscle fiber cells and (B′) blastema mesenchyme (red arrows), and they are associated with basal lamina (green arrows, Laminin) at myosepta (red arrowhead). (C) Summary of Pax3/7 expression during regeneration (pink nuclei). Myoseptal boundaries are indicated by green lines. All images show longitudinal sections with anterior to the left and dorsal to the top. epi, epidermis; no, notochord; nt, neural tube. (Scale bar: 50 μm.)

Fig. 6.

Small and large animals show differences in proliferation and Pax3/7 distribution. (A, A′, B, C, and C′) Proliferative cells labeled with anti-PH3 (green arrows) are present in the blastemas of small (A and A′) and large (C and C′) amphioxus at the same postregenerative stages. Here, 17 dpa blastemas are shown. Posterior is to the upper right. (B) Detail of Pax3/7 protein distribution in proliferating cells (PH3⁺) of a blastema from a small individual. (D) The maximal anteroposterior extent of proliferation and Pax3/7 expression increase with time postamputation and decrease with size of the animal, respectively, using nonparametric statistics (**P ≤ 0.05, ***P ≤ 0.005; n.s. = not significant). In D, Upper, both size classes were pooled. For descriptive purposes, mean values ± SEM are shown. dpa, Days postamputation; epi, epidermis; no, notochord; nt, neural tube; S, small; L, large. (Scale bar: 50 μm.)