Live imaging reveals the progenitors and cell dynamics of limb regeneration

Abstract

Regeneration is a complex and dynamic process, mobilizing diverse cell types and remodelling tissues over long time periods. Tracking cell fate and behaviour during regeneration in active adult animals is especially challenging. Here, we establish continuous live imaging of leg regeneration at single-cell resolution in the crustacean Parhyale hawaiensis. By live recordings encompassing the first 4-5 days after amputation, we capture the cellular events that contribute to wound closure and morphogenesis of regenerating legs with unprecedented resolution and temporal detail. Using these recordings we are able to track cell lineages, to generate fate maps of the blastema and to identify the progenitors of regenerated epidermis. We find that there are no specialized stem cells for the epidermis. Most epidermal cells in the distal part of the leg stump proliferate, acquire new positional values and contribute to new segments in the regenerating leg.

Keywords: Parhyale hawaiensis; developmental biology; live imaging; regeneration; stem cells.

Figure 1.. Imaging leg regeneration in Parhyale hawaiensis.

(A) Amputated Parhyale adult mounted for imaging. The body of the animal is glued onto a coverslip, using a small piece of broken coverslip as a spacer (asterisk). The immobilized leg was amputated as marked with the dashed line. (B) Mounting of the coverslip carrying live Parhyale in a chamber for live imaging (see Materials and methods). (C) Outline of Parhyale thoracic leg (T4 or T5); individual podomeres are highlighted and the position of amputations marked with a dashed line. (D–D’’’) Cellular organization at the distal part of the amputated leg stump. Leg of a mosaic individual expressing H2B-EGFP specifically in the ectoderm (Konstantinides and Averof, 2014); fixed 63 hr post amputation and stained with antibodies for EGFP and acetylated tubulin to reveal ectodermal nuclei and neurons, respectively, and DAPI to label all nuclei. (E) 3-dimensional reconstruction of the same leg stump. (F) Single frame from live recording #04, showing histone-EGFP-labelled nuclei on the leg stump, 52 hr post amputation. Arrowheads and circles mark dividing cells in metaphase and telophase, respectively. (G) Leg stump of a mosaic individual expressing lyn-tdTomato and H2B-EGFP specifically in the mesoderm, 20 hr post amputation. Muscles persist in the proximal part of the leg stump but degenerate in the distal part (top right). The distal part of the leg stump contains a thin strand of interconnected mesodermal cells. DOI: http://dx.doi.org/10.7554/eLife.19766.002

Figure 2.. Overview of timelapse recordings captured by confocal microscopy.

For each recording (numbered #1 to #13) we indicate the time period covered relative to the time of amputation (hpa, hours post amputation), the onset of cell proliferation, molting events and survival. Further details on each recording are given in Table 1. DOI: http://dx.doi.org/10.7554/eLife.19766.013

Figure 3.. Phases of Parhyale leg regeneration.

Three phases of regeneration are defined based on distinct cellular events and behaviours observed by live imaging (see text). Immediately after amputation, haemocytes adhere to the wound surface and close the wound (phase 1). In the hours that follow, a melanized scab (shown in brown) forms at the site of the wound, surrounding the haemocytes (early phase 2). The leg epithelium (depicted as a thin line surrounding the leg) then closes over the wound, under the surface of the melanized scab. Muscles (shown in red) at the distal part of the limb stump usually detach and degenerate, while those in proximal parts remain intact. During phase 2 we observe very limited or no cell proliferation. Phase 3 is marked by the onset of extensive cell proliferation and cell movements, leading to extensive growth and morphogenesis. This phase results in the formation of an elongated and patterned leg primordium within the amputated limb stump. Mesodermal cells, haemocytes and macrophages (shown in red), as well as nerves (in blue), are present in the inner spaces of the leg stump throughout the regenerative process. DOI: http://dx.doi.org/10.7554/eLife.19766.015

Figure 4.. Wound closure during Parhyale leg regeneration.

(A–A’’) Mosaic Parhyale expressing EGFP in haemocytes show the role of these cells in the early stages of wound closure (same individual as in Video 2). (A) EGFP-labelled haemocytes in the leg prior to amputation; the outline of the leg and the amputation plane are highlighted. (A’) In the first few minutes post amputation we observe bleeding (haemocytes marked by arrowhead) and haemocytes adhering to the wound. (A’’) 15 min post amputation the bleeding has stopped and the wound is plugged by a mass of adhering haemocytes. (B–B’’) Epithelial closure and wound melanization followed live in a transgenic animal expressing H2B-EGFP in all cells (still images from Video 3). (B) Amputated leg prior to epithelial closure and melanization; the amputation plane is marked with a dashed line. (B’) 5 hr post amputation (hpa) a new epithelial layer has formed under the wound (arrowhead); more distally, the mass of haemocytes is not yet melanized (curly bracket). (B’’) 9 hr post amputation, the distal part of the stump has been melanized; the haemocytes are embedded in the melanized scab and their fluorescence is no longer visible. DOI: http://dx.doi.org/10.7554/eLife.19766.016

Figure 5.. Cell proliferation and cell movements during Parhyale leg regeneration.

(A) Cell lineages at the distal leg stump, tracked 48–111 hr post amputation (hpa) in recording #03. Vertical lines depict individual cells of the outer (blue) or inner (in red) cell layers tracked at successive timepoints. Cell divisions are depicted as lineage bifurcations and marked by a black dot. Incomplete lines indicate when a cell was not tracked throughout the recording. (A’) Histogram depicting the number of cell divisions observed at each timepoint. (B–B’’’) Tracks of individual epidermal cells during successive 15-hr time intervals, from recording #03 (Video 5), depicting cell movements at the distal leg stump. The amputation plane is at the top right corner of each panel. Each cell track is colour-coded such that the position of each cell at the start and the end of the given time interval are depicted in blue and red, respectively. (B, B’) Before the onset of extensive cell proliferation (48–78 hpa) cells show limited movements towards the wound surface or no movement. (B’’–B’’’) After the start of cell proliferation (78–108 hpa) epidermal cells participate in extensive morphogenetic movements. The length of the coloured bars corresponds to 50 microns. DOI: http://dx.doi.org/10.7554/eLife.19766.017

Figure 6.. Epidermal cell clones and fate maps in regenerating leg stumps.

(A–C) Tracked cells and their clonal progeny are shown from three separate recordings, #03, #04 and #13, respectively. The illustrations depict the outline of the regenerating leg epithelium (grey line) at the start (left) and the end (right) of cell tracking. Individual nuclei and their clonal progeny are colour coded. The leg stumps are shown with their distal end to the right and ventral side down. The outline of the surrounding exoskeleton is depicted by a black line. (A’–C’) Same tracking data, colour-coded according to the proximo-distal location of each nucleus at the start of cell tracking. In panel 6B, two podomeres, the carpus (Ca) and the propodus (Pr), are labelled. DOI: http://dx.doi.org/10.7554/eLife.19766.018

Figure 7.. Morphogenesis of regenerating Parhyale legs.

(A) Orientation of cell divisions occurring during the proliferative phase in recording #04 (46–110 hr post amputation). Each point represents a cell division; divisions occurring along the direction of the proximo-distal axis of the leg are at 0˚. (B) Number of cell divisions occurring in successive 2-hr time intervals, in the same dataset (recording #04). (C) Overall shape of regenerating leg before and after elongation (from recording #04). The outer black line shows the exoskeleton of the amputated leg that constrains the regenerating leg. Arrows highlight the detachment of the epidermis from the exoskeleton and the retraction of the leg proximally. Coloured circles depict the proximal movement of four cell clones (taken from Figure 6B) in the proximal part of the blastema. DOI: http://dx.doi.org/10.7554/eLife.19766.019