Fig. 1. Bulky skeletal elements in the regenerating Pleurodeles waltl limb.

A Micro-CT scans and segmented 3D models of postmetamorphic Pleurodeles waltl limbs with depicted slice planes showing the original CT slice (left), Avizo segmentation, i.e. non-smoothed after manual segmentation, (middle) and after smoothing, color-coded model from VG studio (right). The segmentation steps are described in detail in⁶⁰. (upper) Uninjured limb, (middle) Regenerating limb, 5 w.p.a., (bottom) Regenerated limb, 50 w.p.a. Scale bars 1 mm. B Amputated limbs in post-metamorphic Pleurodeles waltl were analyzed at 2, 3, 4, 5, 6 and 50 weeks post-amputation (w.p.a.). Note the regenerating limb’s skeletal elements consist of cartilage during the patterning and outgrowth phases of limb regeneration. Ossification did not appear until the limb reached the contralateral control’s approximate size and anatomical composition. Blue arrows highlight the increased thickness found in the regenerated bones compared to contralateral control limbs. Scale bars, 1 mm. C Quantification of the volumes occupied by ossified bone in the radius and ulna of fully regenerated (50w.p.a.) versus control limbs of adult Pleurodeles waltl. Regenerated limbs contained more volume of bone than uninjured controls (2-way ANOVA: n.s. Interaction, p = 0.9443; n.s. Zeugopodial element, p = 0.7773; * Regeneration, P = 0.0153). n = 3 limbs per condition. Data are presented as mean values + /- SEM. D Scheme of experimental design and location of amputation in larval Pleurodeles waltl. E Alcian blue / Von Kossa staining highlights the skeletal elements of the contralateral control humerus at 37 days post-amputation (d.p.a.). White arrow points to gap in the mineralisation, and black arrows point to the highly mineralised bone. Scale bar, 200 µm. F Alcian blue / Von Kossa staining highlights the skeletal elements of the regenerating humerus at 37 days post-amputation (d.p.a.). White arrows point to gaps in the mineralisation, and black arrows point to the slightly mineralised bone. Scale bar, 200 µm. G Alcian blue / Von Kossa staining highlights the ossification of the contralateral control humerus at 59 days post-amputation (d.p.a.). Scale bar, 100 µm. H Alcian blue / Von Kossa staining highlights the ossification of the regenerating humerus at 59 days post-amputation (d.p.a.). Scale bar, 100 µm.