Fig. 10
Effect of a heat shock on hsp70 and dnaJb4 expression, cell proliferation, and regeneration of distal fragments. A The experimental design for producing distal and proximal fragments by bisection. The proximal fragment regenerates distal structures, but the distal fragment does not regenerate proximal structures. Distal fragments are maintained at 16 °C or subjected to 25 °C. See Fig. 1H for labels. B–G In situ hybridization showing hsp70 or dnaJb4 expression in the branchial sac vasculature (arrowheads) of distal (C, F) or proximal (B, E) fragments cultured at 16 °C and 25 °C heat-shocked distal fragments (D, G). EN, endostyle. Arrows indicate pigmentation in the oral siphon (OS), atrial siphon (AS), and bisection plane (W). DT, digestive tract. Scale bar: 30 μm; same magnification in B–G. H–L EdU labeling. H Quantification of EdU labeling in the branchial sac vasculature of heat-shocked and control distal fragments. EdU quantification areas are shown in Fig. 1A. Asterisk: significance (t = − 5.67, df = 18, p < .00001) by Student’s one-tailed T test. I–L EdU labeling in the OS (I, J) and branchial sac vasculature (arrowheads) (K, L) of a control (I, K) and a heat-shocked (J, L) distal fragment. Scale bars: 10 μm; same magnification in I, J and K, L. E, M–P OS regeneration in heat-shocked distal fragments. M Percentage of OS regeneration in heat-shocked and control distal fragments. The number of distal fragments assayed is shown at the base of each bar. Asterisk: significance (χ2 = 46.4129, p < .000001) by a chi-square test. N A heat-shocked distal fragment showing OS regeneration at 8 days post-amputation. Dashed line: original amputation plane. AS, atrial siphon; W, proximal wound site. Scale bar: 50 μm. O–P OS regeneration in heat-shocked distal fragments at 8 days post-amputation showing O oral siphon pigment organs (OPO) and P phalloidin-labeled circular muscle bands (CMB). Scale bar: 10 μm; same magnification in O, P. Combined data from three or more replicate experiments