Figure 1.
Fin fold organization and regeneration.
A Representation of the composition of the 2 dpf fin fold cell types and structures, including their respective organization. B Schematic overview representing the main findings occurring after amputation of the 2 dpf larva fin fold, as a part of the regenerative process, addressed throughout the manuscript.
Figure 2.
Abrupt tissue contraction and actomyosin cable formation initiate wound healing.
A–D Sequential images of a representative in vivo 55 min time-lapse movie of an Utrophin-GFP mRNA-injected 2 dpf larva at different time-points after amputation. A At 5minpa, there is accumulation of actin at the leading edge cells. B By 8 minpa the actin cable is assembled (arrow) and there is tissue contraction - note displacement of arrowheads in comparison with A. C At 30 minpa, the tissue has fully contracted. D At 60 minpa, the wound inflicted by the amputation is closed. E–H Sequential images of a representative in vivo 55 min time-lapse movie of a 2 dpf actb1:myl12.1-eGFP transgenic larva. E At 5minpa, there is accumulation of myosin at the leading edge cells. F By 8 minpa the myosin cable is assembled (arrow) and there is tissue contraction - note displacement of arrowheads in comparison with E. G At 30 minpa, the tissue has fully contracted. H At 60 minpa, the wound inflicted by the amputation is closed. I–L Sequential images of a representative in vivo 55 min time-lapse movie of a 2 dpf ctnna-Citrine transgenic larva at different time-points after amputation. I At 5minpa, there is accumulation of alpha-catenin at the leading edge cells. J By 8 minpa this accumulation appears to localize to the actomyosin cable (arrow) and there is tissue contraction - note displacement of arrowheads in comparison with I. K At 30 minpa, the tissue has fully contracted and alpha-catenin is still localized at the wound edge. L At 60 minpa, the wound inflicted by the amputation is closed. Anterior is on the left and scale bars correspond to 50 µm in all images; n = 5 larvae per condition.
Figure 3.
Epidermal tissue growth is enhanced upon fin fold amputation.
A Experimental outline of the live imaging procedures taking into account not only the post amputation hours (hpa) but also the developmental days of the larvae (dpf). All the amputations were performed in 2 dpf larvae, and these were allowed to regenerate until the desired hour, the time point at which they were imaged for 5 hours. Taking into account that the regeneration procedure takes several days, age-matched uncut controls were imaged in the same conditions and for the same amount of time for accurate comparison. B Representative maps of vector velocity fields (VVFs) depicting the tissue movement direction along 5 h sequential time-lapse imaging of the fin fold regenerative process and respective age-matched uncut controls of β-actin:mGFP transgenics. C Representative heat maps of the VVFs shown in B depicting the tissue velocity in the fin fold area along the same 5 h sequential blocks of the fin fold regenerative process and respective age-matched uncut controls. Red end of the spectrum correlates with higher velocity (0 to 10 µm.hour−1) within a given experiment. D Average velocity (µm.hour−1) of VVFs of 5 larvae per condition represented in B–C. Color code matches the Experimental Outline in A. *P value<0.05; Mann-Whitney test values: 2 dpf uncut<>3 dpf uncut = 0.03; 2 dpf uncut<>1–6 hpa = 0.03; 3 dpf uncut<>21–26 hpa = 0.03; 3 dpf uncut<>31–36 hpa = 0.03; 4 dpf uncut<>41–46 hpa = 0.03; 4 dpf uncut<>51–56 hpa = 0.02; 5 larvae per condition. Anterior is on the left and scale bars correspond to 50 µm.
Figure 4.
Global levels of proliferation significantly increase during regeneration in a non-spatially restricted manner.
A Representative projections of H2a.f/z-GFP transgenics showing total cell divisions (marked in red) which occurred during 5 hour sequential time-lapse imaging movies of the fin fold regenerative process and respective age-matched uncut controls. B Average number of cell divisions occurring per 100 µm2 in the several conditions represented in A. Color code matches the Experimental Outline in Fig. 3A. **P value<0.01, *P value<0.05; Mann-Whitney test values: 2 dpf uncut<>3 dpf uncut = 0.008; 2 dpf uncut<>4 dpf uncut = 0.008; 2 dpf uncut<>11–16 hpa = 0.008; 3 dpf uncut<>21–26 hpa = 0.02; 3 dpf uncut<>31–36 hpa = 0.06; 5 larvae per condition. Anterior is on the left and scale bars correspond to 50 µm.
Figure 5.
Cell division orientation in the fin fold shows a tendency for randomization predisposition upon amputation.
Distribution of division angles according to the interval of 225–315° and 45–135° (orange) or 315–45° and 135–225° (blue) in the designated regions 1–4 of the fin fold in A 2 dpf uncut control B 2 dpf 1–6 hpa C 2 dpf 11–16 hpa. Predominant growth direction in the regions 1–4 taking into account the orientation of the majority of cell divisions happening in A' 2 dpf uncut control B' 2 dpf 1–6 hpa C' 2 dpf 11–16 hpa. n = 193 divisions in 2 dpf uncut, n = 175 divisions in 2 dpf 1–6 hpa, n = 258 divisions in 2 dpf 11–16 hpa; 5 larvae per condition.
Figure 6.
Mesenchymal cells in the fin fold change shape and migrate distally upon injury.
A–C Sequential images of a representative in vivo 6 h time-lapse movie of a 2 dpf uncut EF1α:mKO2-zCdt1;osteopontin:eGFP transgenic larva. D–F Sequential images of a representative in vivo 6 h time-lapse movie of a 2 dpf 30 minpa EF1α:mKO2-zCdt1;osteopontin:eGFP transgenic larva. G–J Sequential images of a representative in vivo 1h30 time-lapse movie of a 2 dpf 5 minpa EF1α:mKO2-zCdt1;osteopontin:eGFP transgenic larva. Zoom panels highlight osteopontin positive mesenchymal cells in the central area of the fin fold in the respective time point. White dots mark the same cells along time to allow for better visualization and tracking of cell migration. Dashed lines indicate the outline of the notochord and the edge of the amputated fin fold. Scale bars correspond to 50 µm in all images; 3–5 larvae per condition.
Figure 7.
The shape modification of mesenchymal cells lasts throughout and is specific of regeneration.
A,C,E Representative immunostaining with anti-GFP antibody in uncut transgenic osteopontin:eGFP larvae of 3 dpf, 4 dpf and 5 dpf respectively. B,D,F Representative immunostaining with anti-GFP antibody in amputated transgenic osteopontin:eGFP larvae of 3 dpf 1 dpa, 4 dpf 2 dpa and 5 dpf 3 dpa respectively. A–F Representative z-stack projections of the osteopontin:eGFP labeling. A'–F' Representative single frames of the corresponding zoomed area represented by a square in A–F. Merged and single color images of osteopontin:eGFP labeling the mesenchymal cells (anti-GFP, green), actin (phalloidin, red) and nuclei (DAPI, blue), respectively. 5 Larvae per condition. Scale bar corresponds to 50 µm in all images.