Figure 4.

nZRS reporter expression during metamorphosed limb regeneration and autopod patterning. (a) Representative transgenic nZRS reporter newt with an intact limb (dorsal view), n = 9. The white line indicates the mid humerus (stylopod) amputation plane. (b) Following limb amputations, the mCherry reporter signal (white arrowheads) was detected at 2 h post-amputation in the anterior region. At 14 dpa, the reporter signal was not detected. However, during mid-blastema, at 35 dpa, a defined cluster of cells was detected. Solid white line indicates the amputation plane. At 40 dpa, a mCherry+ cluster was localized in the posterior of the late-blastema, ZPA-l. At the palette stage, at 49 dpa, just before digit I regeneration, a mCherry+ cluster was polarized in the posterior region. (c) Negative control, wild-type individuals uninjected with reporter plasmid, n = 3 limbs. (d) Cross section of transgenic ZPA region at 49 dpa, n = 3. (e) Magnification of white box (d) indicating overlap between mCherry+ and Shh+ cells. The yellow line indicates the margin of the WE. White arrowheads indicate mCherry+ cells; yellow arrowheads indicate Shh+ cells. (f) At 58 dpa, reporter expression was localized in digits III and IV. By 65 dpa, a weak signal was detected in the anterior digits I to III, and digit IV showed a reduction of reporter expression. White dashed lines indicates contour of the limb.. A: anterior; D: dorsal; Di: distal; dpa: days post-amputation; D.v.: dorsal view; P: posterior; P.v: posterior view; Vn: ventral; ZPA: zone of polarizing activity; WE: wound epithelium. Scale bars: (a), 0.5 cm; (b,c,f), 1 mm; (d), 0.5 mm; and (e), 50 µm.