Limited dedifferentiation provides replacement tissue during zebrafish fin regeneration

Abstract

Unlike humans, some vertebrate animals are able to completely regenerate damaged appendages and other organs. For example, adult zebrafish will regenerate the complex structure of an amputated caudal fin to a degree that the original and replacement fins are indistinguishable. The blastema, a mass of cells that uniquely forms following appendage amputation in regenerating animals, is the major source of regenerated tissue. However, the cell lineage(s) that contribute to the blastema and their ultimate contribution(s) to the regenerated fin have not been definitively characterized. It has been suggested that cells near the amputation site dedifferentiate forming multipotent progenitors that populate the blastema and then give rise to multiple cell types of the regenerated fin. Other studies propose that blastema cells are non-uniform populations that remain restricted in their potential to contribute to different cell lineages. We tested these models by using inducible Cre-lox technology to generate adult zebrafish with distinct, isolated groups of genetically labeled cells within the caudal fin. We then tracked populations of several cell types over the entire course of fin regeneration in individual animals. We found no evidence for the existence of multipotent progenitors. Instead, multiple cell types, including epidermal cells, intra-ray fibroblasts, and osteoblasts, contribute to the newly regenerated tissue while remaining highly restricted with respect to their developmental identity. Our studies further demonstrate that the regenerating fin consists of many repeating blastema "units" dedicated to each fin ray. These blastemas each have an organized structure of lineage restricted, dedifferentiated cells that cooperate to regenerate the caudal fin.

Figure 1

A tamoxifen-inducible Cre/lox method for mosaic cell labeling and lineage tracing in the adult zebrafish caudal fin. (A) Embryos carrying both dusp6:Cre-ERT2 and EAB:EGFP-FlEx-mCherry transgenes are briefly exposed to tamoxifen to sporadically induce rare genetic recombination events that permanently switch those cells and their descendants to mCherry from EGFP expression. (B, C) Whole mount epifluorescent images of 3 day old Tg(dusp6:Cre-ERT2, EAB:EGFP-FlEx-mCherry) fish that were treated with tamoxifen (1 µM) at 30% epiboly for 48 hours. Mosaic mCherry⁺ cells (magenta) are observed in various tissues (B), including pectoral fin mesenchyme (C). The white arrow highlights mCherry⁺ cells. (D, E) The EAB:EGFP-FlEx-mCherry transgene is expressed in various cell lineages that make up the adult caudal fin. Longitudinal (C) and transverse (D) sections of adult caudal fins of Tg(EAB:EGFP-FlEx-mCherry) animals. EGFP expressing cells (green) were are stained with Hoechst to visualize nuclei (magenta). (F–I, F*–I*) Tg(dusp6:Cre-ERT2, EAB:EGFP-FlEx-mCherry) adult animals, treated as above, exhibit spatially restricted mCherry⁺ mosaics in four distinct classes. The dashed box marked with an asterisk represents the region shown at higher magnification in the panels directly below (F*–I*). (F and F*) Class 1 epidermal mosaics. (G and G*) Class 2 fibroblast mosaics. (H and H*) Class 3 osteoblast mosaics. (I and I*) Class 4 putative macrophage mosaics. All images show overlaid EGFP (green) and mCherry (magenta) expression. The top and bottom of each panel corresponds to the distal and proximal regions of the fin, respectively.

Figure 2

Newly regenerated epidermis is derived from pre-existing epidermal cells. (A–D, A*–D*) Whole mount epifluorescent images from the caudal fin from a Tg(dusp6:Cre-ERT2, EAB:EGFP-FlEx-mCherry) animal containing Class 1 labeled epidermal cells before amputation (A, A*), 1 dpa (B, B*), 4 dpa (C, C*), and 14 dpa (D, D*). Dashed boxes marked with an asterisk represent the region shown at higher magnification in the panel directly below. (A–D) and (A*–D*) are images acquired at 25× and 120× magnification, respectively. EGFP⁺ cells are in green and mCherry⁺ cells are pseudocolored magenta. The top and bottom of each panel corresponds to the distal and proximal regions of the fin, respectively. The dashed yellow line shows the approximate amputation site and the white arrows point to epidermal cells found laterally to the starting population.

Figure 3

Epidermal cells do not contribute to the blastema or change fate during regeneration. (A–L) Longitudinal sections of the caudal fin of Tg(dusp6:Cre-ERT2, EAB:EGFP-FlEx-mCherry) Class 1 mosaic animals demonstrating mCherry expression (red, A, E, I) and stained with anti-p63 antibodies to mark epidermal cells (green, B, F, J) and with Hoechst to mark nuclei (blue, C, G, K). The three-color overlays are shown in (D, H, L). (A–D) mCherry⁺ epidermal cells in Tg(dusp6:Cre-ERT2, EAB:EGFP-FlEx-mCherry) Class 1 mosaic animals remain positive for the epidermal maker p63 at 2 dpa. The yellow dashed line marks the approximate amputation site and the white dashed line marks the boundary between the epidermis and the blastema. (E–L) Stained fin sections from the same Tg(dusp6:Cre-ERT2, EAB:EGFP-FlEx-mCherry) Class 1 mosaic animal prior to amputation (E–H) and 7 dpa (I–L). White arrows indicate mCherry⁺/p63⁺ epidermis.

Figure 4

Intra-ray fibroblasts in regenerated fins are derived from pre-existing intra-ray fibroblasts. (A–D, A*–D*) Whole mount epifluorescent images of a Class 2 intra-ray fibroblast mosaic labeled caudal fin of the same Tg(dusp6:Cre-ERT2, EAB:EGFP-FlEx-mCherry) animal before amputation (A, A*), 1 dpa (B, B*), 4 dpa (C, C*), and 14 dpa (D, D*). The dashed box marks the zoomed region in the panel directly below. mCherry⁺ cells are shown in magenta. All other cells are EGFP⁺ (green). The amputation plane is shown with a dashed yellow line.

Figure 5

Intra-ray fibroblasts are a proliferating component of the blastema. (A–L) Longitudinal sections of Class 2 mosaic fins from Tg(dusp6:Cre-ERT2, EAB:EGFP-FlEx-mCherry) harvested 2 dpa and monitored for mCherry expression (A, E, I, red) and immunostained with indicated antibodies (B, F, J, green). Nuclei are stained with Hoechst (C, G, K, blue), and the three-color overlay is shown in each case (D, H, L). (A–D) Cells derived from intra-ray fibroblasts populate the blastema at 2 dpa. The section shows mCherry expression (A, red) and is immunostained with anti-tenascin-C (tenC) antibodies (B, green). The white arrow indicates mCherry⁺ intra-ray fibroblasts and the white dashed line denotes the boundary between epidermis and blastema. (E–H) mCherry⁺ intra-ray fibroblasts do not express markers for osteoblasts in regenerating tissue. mCherry⁺ cells are shown in red and zns-5 antibodies detect osteoblasts (F, green). White arrows indicate mCherry⁺ cells in the blastema that do not co-localize with zns-5⁺ osteoblasts (yellow arrow). The white dashed line indicates the boundary between epidermis and blastema. (I–L) Intra-ray fibroblasts are a source of proliferating cells in the blastema. A 2 dpa section co-stained with anti-dsRed antibodies to detect mCherry⁺ cells (I, red) and anti-PCNA antibodies to detect proliferating cells (J, green). The white arrows indicate mCherry⁺/PCNA⁺ intra-ray fibroblasts in the blastema. The yellow arrow marks a mcherry⁺/PCNA⁻ intra-ray fibroblast, and the border between epidermis and blastema is marked with a white dashed line.

Figure 6

Newly regenerated bone is formed by pre-existing osteoblasts. (A–D, A*–D*) Whole mount images of a Tg(dusp6:Cre-ERT2, EAB:EGFP-FlEx-mCherry) Class 3 mosaic caudal fin containing mCherry labeled osteoblasts before amputation (A, A*), 1 dpa (B, B*), 4 dpa (C, C*), and 14 dpa (D, D*). The dashed box denotes the region magnified in the panel directly below. All cells are EGFP⁺ (green), except mCherry⁺ genetically recombined cells and their descendants (magenta). White arrows mark osteoblasts and yellow arrows denote cells co-labeled with mCherry⁺ osteoblasts.

Figure 7

Osteoblasts in the caudal fin populate the blastema but remain fate restricted. (A–L) Longitudinal sections of the caudal fin of Tg(dusp6:Cre-ERT2, EAB:EGFP-FlEx-mCherry) Class 3 mosaic animals showing mCherry expression in osteoblasts (A, E, I, M, red) and stained with indicated antibodies (B, F, J, N, green) and with Hoechst to mark nuclei (C, G, K, O, blue). The three-color overlays are shown in (D, H, L, P). (A–D) Osteoblasts contribute to the blastema. A fin section observed for mCherry expression and immunostained with zn-3 antibodies to mark osteoblasts. White arrows indicate mCherry⁺ cells (red) in the blastema that co-localize with zn-3⁺ osteoblasts (green). The dashed yellow line marks the amputation site and the dashed white line indicates the epidermis-blastema boundary. (E–H) Osteoblast-derived blastema cells proliferate. A fin section from the same animal in (A–D) stained with anti-dsRed antibodies to detect mCherry expression (red) and with anti-PCNA antibodies (green) to mark proliferating cells. White arrows indicate mCherry⁺/PCNA⁺ intra-ray fibroblasts in the blastema. The border between epidermis and blastema is marked with a white dashed line. (I–P) Osteoblasts do not change fate during regeneration. Stained fin sections from the same Tg(dusp6:Cre-ERT2, EAB:EGFP-FlEx-mCherry) Class 3 mosaic animal prior to amputation (I–L) and 7 dpa (M–P). mCherry expression is red and anti-zn-3 antibody staining marks osteoblasts in green. The white arrows mark cells that are both mCherry and zn-3 positive.

Figure 8

Osteoblasts dedifferentiate within the blastema. (A–D, H–K) Longitudinal sections from a Tg(sp7:EGFP) animal 2 dpa stained with zn-3 (A–D) or zns-5 antibodies (H–K). White arrows point to EGFP⁺/zn-3⁺ (or zns-5⁺) immature, dedifferentiated osteoblasts and yellow arrows point to EGFP⁺/zn-3⁻ (or zns-5⁻) mature, differentiated osteoblasts. EGFP signal is shown in green, zn-3⁺ (B, D), or zns-5⁺ (I, K) cells are red, and Hoechst-stained nuclei are blue. The border between epidermis and blastema is marked with a white dashed line. (E–G, L–M) Relative signal intensity levels for EGFP (E, L), zn-3 or zns-5 antibody staining (F, M), and Hoechst (G, N). Fluorescence intensity levels (z-axis) within the indicated rectangle (thin dashed yellow line) are plotted as a 3-dimensional surface using Image J software. The amputation site is marked with an arrowhead and the proximal and distal regions relative to the amputation site are indicated. The white dashed line indicates the border between epidermis and blastema.

Figure 9

A restricted cell lineage model for fin regeneration. During homeostasis, the caudal fin is composed of various lineage restricted differentiated cells existing in a “ground state”. Fin amputation initiates wound healing by a motile epidermis and results in the conversion of ground state cells to “transition state” cells by the process of limited dedifferentiation. Transition state cells, including those derived from osteoblasts and intra-ray fibroblasts, contribute to an organized proliferating regenerative blastema capable of outgrowth and re-differentiation. The newly regenerated tissue is derived from transition state cells that differentiate into cells only of the same lineage, returning to their ground state as the fin reforms.