The art of fin regeneration in zebrafish

Abstract

The zebrafish fin provides a valuable model to study the epimorphic type of regeneration, whereby the amputated part of the appendage is nearly perfectly replaced. To accomplish fin regeneration, two reciprocally interacting domains need to be established at the injury site, namely a wound epithelium and a blastema. The wound epithelium provides a supporting niche for the blastema, which contains mesenchyme-derived progenitor cells for the regenerate. The fate of blastemal daughter cells depends on their relative position with respect to the fin margin. The apical compartment of the outgrowth maintains its undifferentiated character, whereas the proximal descendants of the blastema progressively switch from the proliferation program to the morphogenesis program. A delicate balance between self-renewal and differentiation has to be continuously adjusted during the course of regeneration. This review summarizes the current knowledge about the cellular and molecular mechanisms of blastema formation, and discusses several studies related to the regulation of growth and morphogenesis during fin regeneration. A wide range of canonical signaling pathways has been implicated during the establishment and maintenance of the blastema. Epigenetic mechanisms play a crucial role in the regulation of cellular plasticity during the transition between differentiation states. Ion fluxes, gap-junctional communication and protein phosphatase activity have been shown to coordinate proliferation and tissue patterning in the caudal fin. The identification of the downstream targets of the fin regeneration signals and the discovery of mechanisms integrating the variety of input pathways represent exciting future aims in this fascinating field of research.

Keywords: blastema; caudal fin; epigenetics; regeneration; zebrafish.

Figure 1

The skeleton of the zebrafish caudal fin. (A)−(D) Whole mount view of an adult caudal fin stained with Alcian Blue and Alizarin Red to visualize the skeleton. (A) The bi‐lobed morphology of the caudal fin fold is stabilized by 16−18 segmented and occasionally bifurcated bony rays (stained structures), named lepidotrichia, that are interconnected by soft interray tissue (unstained regions between the bones). The segmental borders contain ligaments with a regular spacing along the proximo‐distal axis (a whitish ladder‐like pattern of each ray). The bones are predominantly composed of calcified matrix (magenta), with the exception of the distal parts which remain non‐mineralized (cyan). (B) A higher magnification of the distal region shows a gradual decrease of the calcification level towards the fin margin. The length of segments is nearly identical in proximal (magenta) and distal (cyan) parts of the rays. (C) The tips of the rays are supported by a brush‐like bundle of fine spicules, called actinotrichia, which surround the apical‐most segment of the lepidotrichia and expand further distally beyond the end of the bone. (D) The proximal segments of the rays are at least three times broader than the distal calcified segments (compared with B), but their length remains nearly constant. Scale bars: (A) 1000 μm; (B)−(D) 100 μm.

Figure 2

The histological organization of an uninjured and regenerating caudal fin. (A) Schematic representation of the fin structure with the planes of sectioning along the interray (green frame) and rays (blue frame). (B)−(I) Longitudinal fin sections stained with hematoxylin and eosin. (B) Each lepidotrichium consists of a pair of concave bones (b) that appear as parallel rods underneath the multilayered epidermis (e). Bones are tightly covered by flattened osteoblasts that deposit the bone matrix. The mesenchymal tissue (m) between the bones is composed of connective tissue containing densely interconnected fibroblasts, nerves, and arteries (a). (C) The interray is devoid of skeletal elements and contains loose connective tissue. (D) At 30 hpa, the blastema (bl) appears as a cluster of undifferentiated mesenchymal cells covered by a wound epidermis (we) above the amputation plane (white dashed line). Blastema formation results from the dedifferentiation of cells located in the stump that progressively lose their specialized morphology, initiate proliferation, and migrate distally toward the amputation plane. (E) At 72 hpa, the blastemal outgrowth exhibits a spatial organization of the newly formed tissue. (F) Higher magnification of the distal part of the outgrowth (apical signaling zone with slowly cycling cells). Mesenchymal cells become elongated perpendicularly to the growth (proximo‐distal) axis. The basal layer of the wound epithelium (bwe) contains columnar cells. (G) Higher magnification of the proximal part of the outgrowth (proliferation and redifferentiation zone). Dedifferentiated osteoblasts (ob) are tightly interconnected and remain aligned underneath the wound epidermis. The basal layer of the wound epithelium (bwe) contains cuboidal cells. The mesenchymal cells are round and loosely distributed. Scale bars: 50 μm.

Figure 3

The regeneration process of the caudal fin in zebrafish. (A) Time‐lapse imaging of the same fin during the regeneration process at 27°C. Uncut, the original fin prior to amputation presents a bi‐lobed morphology. At 1 dpa, white tissue above the amputation consists of the wound epidermis and a few blastema cells. At 3 dpa, a white excrescence above the amputation plane contains the blastema, which, despite its uniform appearance, exhibits subdivisions at the cellular and molecular level. At 6 dpa, the outgrowth extends very rapidly; the white tissue is maintained at the fin margin, while the proximal outgrowth starts to display bone structures and pigmentation, which are the macroscopic markers of tissue redifferentiation. At 12 dpa, fin regeneration is at its advanced stage. At 20 dpa, the size of the fin nearly reaches its original size and pattern. The white margin of tissue remains at the tip for homeostatic growth/regeneration. (B) Higher magnifications of the fin surface at the position of amputation (white dashed line) at the respective time points are indicated in the upper panel (A). (C) The milestones of the fin regeneration process. Scale bars: (A) 1000 μm; (B) 200 μm.