Figure 1: Live imaging of cartilage growth.

(a) The growth plate cartilage displays a stem-loop shape and contains four zones along the PDA: resting (RZ), proliferative (PZ), prehypertrophic (PHZ) and hypertrophic (HZ) zones. (b–d) Schematic diagrams of metacarpal culture and imaging. (b) Chick forelimb bud was injected with RCAS-GFP and the viral-infected metacarpal was dissected for organ culture. (c) A mold (grey) was inserted into molten agarose on an imaging dish to create grooves (d), which were used to hold the metacarpals stably for imaging through the objective (light blue) on an inverted laser scanning microscope. (e) Top view of the metacarpal sitting in the groove. (f) The same metacarpal exhibiting similar extension on both sides after live imaging for 55 h. The red circles in (e) and (f) depict the region imaged. (g) Two orthogonal sections shown in the 3D rendered image of the GFP-expressing (green) metacarpal at t=29 h (chosen for the best depiction) of live imaging. The white cuboid drawn shows the approximate region selected for the PZ cell tracking. (h) Side view of the same sample. The middle 90-μm region spanning both stem and loop parts was digitally sliced into three sections along the imaging axis (Methods) to facilitate cell segmentation. Scale bars, (e,f) 500 μm, (g) 50 μm and (h) 20 μm.